P-phenylene ethynylene compounds as bioactive and detection agents

ABSTRACT

Various embodiments relate to p-phenylene ethynylene compounds as bioactive and detection agents. In various embodiments, the present invention provides a method of inducing germination of microbial spores including contacting the microbial spores with a p-phenylene ethynylene compound. In various embodiments, the present invention provides a method for detecting an enzyme, a method of protein analysis, or a method of detecting a chemical agent, including introducing a p-phenylene ethylylene compound to a composition including an enzyme substrate, and analyzing the fluorescence of the p-phenylene ethynylene compound. Various embodiments provide sensors that include a p-phenylene ethynylene compound and an enzyme substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S.Provisional Application No. 61/953,311, filed on Mar. 14, 2014, U.S.Provisional Application No. 61/954,923, filed on Mar. 18, 2014, U.S.Provisional Application No. 61/955,522, filed on Mar. 19, 2014, and U.S.Provisional Application No. 62/012,780, filed on Jun. 16, 2014, thedisclosures of which are incorporated by reference herein in theirentireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under HDTRA-11-1-0004awarded by the Defense Threat Reduction Agency (DTRA), under DMR1207362and CBET-1150855 awarded by the National Science Foundation (NSF), andunder R21 NS07708 awarded by the National Institutes of Health (NIH).The U.S. Government has certain rights in this invention.

BACKGROUND

Agents that can kill the viable bacteria, such as antibiotics, can beineffective to terminate the viability of the bacterial spores, makingpopulations of such bacteria difficult to control. Even if the entiremature population is killed, viable spores are still available forgermination to restore a potentially pathogenic bacterial population.

Detecting enzymes, proteins, or chemical agents can be valuable for awide variety of uses. For example, abnormal formation and deposition ofamyloid protein aggregates is associated with a number ofneurodegenerative diseases, including, but not limited to, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, systemic amyloidosisand inherited organ-specific amyloidoses, and transmissible priondiseases such as bovine spongiform encephalopathy, chronic wastingdisease, and sheep scrapie. Each of these diseases is characterized bysymptoms including cross-β-sheet rich aggregates, formed fromcharacteristic proteins depending upon the specific disease.Understanding, diagnosing, and treating these diseases require tools tolocate and track the formation of amyloid aggregates in livingorganisms, particularly the putative toxic aggregate forms. The primarymethod for amyloid detection is histopathological staining of tissuesections with fluorescent dyes, of which the commonest currently isThioflavin T. Existing dyes have limitations; they target primarilymature aggregates and they cannot distinguish between amyloids withdiffering conformations, particularly oligomeric/pre-fribillaraggregates that are considered the primary toxic species.

SUMMARY OF THE INVENTION

In various embodiments, the present invention provides a method ofinducing germination of microbial spores including contacting themicrobial spores with a p-phenylene ethynylene compound.

In various embodiments, the present invention provides a method fordetecting an enzyme. The method includes (i) introducing an enzyme to acomposition including a p-phenylene ethynylene compound and an enzymesubstrate. The method also includes (ii) analyzing the change influorescence of the p-phenylene ethynylene compound followingintroduction of the enzyme.

In various embodiments, the present invention provides a method fordetecting an enzyme. The method includes (i) introducing an enzyme to acomposition including a p-phenylene ethynylene compound and an enzymesubstrate; and (ii) analyzing the change in fluorescence of thep-phenylene ethynylene compound following introduction of the enzyme.The p-phenylene ethynylene compound has the structure:

The variable s is about 1 to about 3. The enzyme substrate is1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol). The enzyme is atleast one of phospholipase A1, phospholipase A2, and phospholipase C.

In various embodiments, the present invention provides a method fordetecting an enzyme. The method includes (i) introducing an enzyme to acomposition including a p-phenylene ethynylene compound and an enzymesubstrate. The method also includes (ii) analyzing the change influorescence of the p-phenylene ethynylene compound followingintroduction of the enzyme. The p-phenylene ethynylene compound has thestructure:

The variable t is about 1 to about 3. The enzyme substrate is lauroylcholine. The enzyme is acetylcholinesterase.

In various embodiments, the present invention provides a sensor. Thesensor includes a p-phenylene ethynylene compound and an enzymesubstrate.

In various embodiments, the present invention provides a sensor. Thesensor includes a cationic p-phenylene ethynylene compound and ananionic enzyme substrate. The p-phenylene ethynylene compound has thestructure:

The variable s is about 1 to about 3. The anionic enzyme substrate is1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

In various embodiments, the present invention provides a sensor. Thesensor includes a p-phenylene ethynylene compound and an enzymesubstrate. The p-phenylene ethynylene compound has the structure:

The variable t is about 1 to about 3. The enzyme substrate is lauroylcholine.

In various embodiments, the present invention provides a method forprotein analysis. The method includes (i) introducing a p-phenyleneethynylene compound to a biological sample including at least oneprotein. The method also includes (ii) analyzing the fluorescence of thep-phenylene ethynylene compound in the presence of the biological sampleincluding the at least one protein.

In various embodiments, the present invention provides a method forprotein analysis. The method includes (i) analyzing the fluorescence ofa p-phenylene ethynylene compound. The method includes (ii) introducingthe p-phenylene ethynylene compound to a biological sample including atleast one protein. The method includes (iii) analyzing the fluorescenceof the p-phenylene ethynylene compound in the presence of the biologicalsample including the at least one protein. The method also includes (iv)determining the morphology of the at least one protein in the biologicalsample by analyzing spectral changes between the fluorescence of thep-phenylene ethynylene compound of step (i) and the fluorescence of thep-phenylene ethynylene compound in the presence of the biological sampleincluding the at least one protein of step (iii). The p-phenyleneethynylene compound has the structure:

The variable s is 1. The protein is at least one of an amyloid betaprotein, Aβ-40, Aβ-42, tau, and α-synuclein, islet amyloid precursorprotein, Huntingtin, prion, lysozyme, TDP-43 (transactive responseDNA-binding protein 43), FUS (fused in sarcoma), and insulin.

In various embodiments, the present invention provides a method fordetecting a chemical agent. The method includes (i) exposing a sensorcomposition including a complex including a p-phenylene ethynylenecompound and an enzyme substrate to a sample. The method includes (ii)introducing an enzyme to the sensor composition of step (i). The methodalso includes (iii) analyzing the change in fluorescence of thep-phenylene ethynylene compound between the exposing step (i) and theintroducing an enzyme step (ii).

In various embodiments, the present invention provides a method fordetecting a chemical agent. The method includes (i) exposing a sensorcomposition including a complex including a p-phenylene ethynylenecompound and an enzyme substrate to a sample. The method includes (ii)introducing an enzyme to the sensor composition of step (i). The methodalso includes (iii) analyzing the change in fluorescence of thep-phenylene ethynylene compound between the exposing step (i) and theintroducing an enzyme step (ii). The p-phenylene ethynylene compound hasthe structure:

The variable t is about 1 to about 3. The enzyme substrate is lauroylcholine. A change in fluorescence between the exposing step (i) and theintroducing an enzyme step (ii) indicates the presence of a chemicalagent that does interact with the enzyme.

In various embodiments, the present invention provides a sensor fordetecting the presence of a chemical agent. The sensor includes a sensorcomposition including a complex including a p-phenylene ethynylenecompound and an enzyme substrate.

In various embodiments, the present invention provides a sensor fordetecting the presence of a chemical agent. The sensor includes a sensorcomposition including a complex including a p-phenylene ethynylenecompound and an enzyme substrate. The p-phenylene ethynylene compoundhas the structure

The variable t is about 1 to about 3. The enzyme substrate is lauroylcholine.

BRIEF DESCRIPTION OF THE FIGURES

The drawings illustrate generally, by way of example, but not by way oflimitation, various embodiments discussed in the present document.

FIGS. 1A-D illustrate flow cytometry-reported fluorescence of stained B.atrophaeus vegetative cells following various treatment conditions, inaccordance with various embodiments.

FIGS. 2A-D illustrate flow cytometry-reported germination of B.atrophaeus spores under various treatment conditions, in accordance withvarious embodiments.

FIGS. 3A-E illustrate scanning electron microscope images of B.atrophaeus spores and vegetative cells, in accordance with variousembodiments.

FIG. 4 illustrates B. anthracis Sterne vegetative cell viability withand without exposure to a PE, in accordance with various embodiments.

FIG. 5 illustrates B. anthracis Sterne spore and germinated vegetativecell viability as a function of starting concentration, in accordancewith various embodiments.

FIGS. 6A-D illustrate absorbance and fluorescence of various p-phenyleneethynylene compounds (PEs) versus wavelength in the presence of varyingamounts of 1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG)or lauroyl choline (LaCh), in accordance with various embodiments.

FIGS. 7A-B illustrate integrated fluorescence versus absorbance forvarious PEs, in accordance with various embodiments.

FIGS. 8A-D illustrate fluorescence versus time for PE with variousconcentrations of DLPG and PLA1 or PLA2, in accordance with variousembodiments.

FIGS. 9A-C illustrate absorbance or fluorescence versus substrateconcentration for various sensors, in accordance with variousembodiments.

FIGS. 10A-B illustrate loss of fluorescence over time and velocityversus substrate concentration, in accordance with various embodiments.

FIGS. 11A-B illustrate absorbance and fluorescence versus time afteraddition of various concentrations of substrate, in accordance withvarious embodiments.

FIGS. 12A-C illustrate fluorescence versus time for a PE/LaCh sensorover time with and without inhibitor, in accordance with variousembodiments.

FIGS. 13A-B illustrate absorbance and fluorescence versus wavelength fora PE alone, with LaCh, and with various inhibitors, in accordance withvarious embodiments.

FIGS. 14A-B illustrate absorbance and fluorescence of a PE with variousconcentrations of DLPG, in accordance with various embodiments.

FIG. 15 illustrates fluorescence enhancement versus hen egg whitelysozyme (HEWL) incubation time, in accordance with various embodiments.

FIG. 16 illustrates mean molar ellipticity versus wavelength for HEWLincubated for various times, in accordance with various embodiments, inaccordance with various embodiments.

FIG. 17 illustrates transmission electron spectroscopy and atomic forcemicroscopy images of HEWL incubated for various times, in accordancewith various embodiments.

FIGS. 18A-H illustrate fluorescence versus wavelength for various PEs,in accordance with various embodiments.

FIG. 19 illustrates fluorescence versus PE concentration in the presenceof HEWL monomers, in accordance with various embodiments.

FIG. 20 illustrates HEWL/PE Förster resonance energy transfer (FRET)efficiencies for various PEs calculated from spectral data, inaccordance with various embodiments.

FIG. 21 illustrates modes of interaction between PEs and HEWL monomersand amyloids, in accordance with various embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of thedisclosed subject matter, examples of which are illustrated in part inthe accompanying drawings. While the disclosed subject matter will bedescribed in conjunction with the enumerated claims, it will beunderstood that the exemplified subject matter is not intended to limitthe claims to the disclosed subject matter.

Throughout this document, values expressed in a range format should beinterpreted in a flexible manner to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. For example, a range of “about 0.1% to about 5%” or “about 0.1%to 5%” should be interpreted to include not just about 0.1% to about 5%,but also the individual values (e.g., 1%, 2%, 3%, and 4%) and thesub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within theindicated range. The statement “about X to Y” has the same meaning as“about X to about Y,” unless indicated otherwise. Likewise, thestatement “about X, Y, or about Z” has the same meaning as “about X,about Y, or about Z,” unless indicated otherwise.

In this document, the terms “a,” “an” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.The statement “at least one of A and B” has the same meaning as “A, B,or A and B.” In addition, it is to be understood that the phraseology orterminology employed herein, and not otherwise defined, is for thepurpose of description only and not of limitation. Any use of sectionheadings is intended to aid reading of the document and is not to beinterpreted as limiting; information that is relevant to a sectionheading may occur within or outside of that particular section. A commacan be used as a delimiter or digit group separator to the left or rightof a decimal mark; for example, “0.000,1” is equivalent to “0.0001.”

In the methods of manufacturing described herein, the acts can becarried out in any order without departing from the principles of theinvention, except when a temporal or operational sequence is explicitlyrecited. Furthermore, specified acts can be carried out concurrentlyunless explicit claim language recites that they be carried outseparately. For example, a claimed act of doing X and a claimed act ofdoing Y can be conducted simultaneously within a single operation, andthe resulting process will fall within the literal scope of the claimedprocess.

The term “about” as used herein can allow for a degree of variability ina value or range, for example, within 10%, within 5%, or within 1% of astated value or of a stated limit of a range, and includes the exactstated value or range.

The term “substantially” as used herein refers to a majority of ormostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or100%.

The term “organic group” as used herein refers to any carbon-containingfunctional group. For example, an oxygen-containing group such as analkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group, acarboxyl group including a carboxylic acid, carboxylate, and acarboxylate ester; a sulfur-containing group such as an alkyl and arylsulfide group; and other heteroatom-containing groups. Non-limitingexamples of organic groups include OR, OOR, OC(O)N(R)₂, CN, CF₃, OCF₃,R, C(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, SO₂R, SO₂N(R)₂,SO₃R, C(O)R, C(O)C(O)R, C(O)CH₂C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂,OC(O)N(R)₂, C(S)N(R)₂, (CH₂)₀₋₂N(R)C(O)R, (CH₂)₀₋₂N(R)N(R)₂,N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂,N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂,N(COR)COR, N(OR)R, C(═NH)N(R)₂, C(O)N(OR)R, C(═NOR)R, and substituted orunsubstituted (C₁-C₁₀₀)hydrocarbyl, wherein R can be hydrogen (inexamples that include other carbon atoms) or a carbon-based moiety, andwherein the carbon-based moiety can be substituted or unsubstituted.

The term “substituted” as used herein in conjunction with a molecule oran organic group as defined herein refers to the state in which one ormore hydrogen atoms contained therein are replaced by one or morenon-hydrogen atoms. The term “functional group” or “substituent” as usedherein refers to a group that can be or is substituted onto a moleculeor onto an organic group. Examples of substituents or functional groupsinclude, but are not limited to, a halogen (e.g., F, Cl, Br, and I); anoxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxygroups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groupsincluding carboxylic acids, carboxylates, and carboxylate esters; asulfur atom in groups such as thiol groups, alkyl and aryl sulfidegroups, sulfoxide groups, sulfone groups, sulfonyl groups, andsulfonamide groups; a nitrogen atom in groups such as amines,hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, andenamines; and other heteroatoms in various other groups. Non-limitingexamples of substituents that can be bonded to a substituted carbon (orother) atom include F, Cl, Br, I, OR, OC(O)N(R)₂, CN, NO, NO₂, ONO₂,azido, CF₃, OCF₃, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy,ethylenedioxy, N(R)₂, SR, SOR, SO₂R, SO₂N(R)₂, SO₃R, C(O)R, C(O)C(O)R,C(O)CH₂C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂, OC(O)N(R)₂, C(S)N(R)₂,(CH₂)₀₋₂N(R)C(O)R, (CH₂)₀₋₂N(R)N(R)₂, N(R)N(R)C(O)R, N(R)N(R)C(O)OR,N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂, N(R)C(O)OR, N(R)C(O)R,N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(═NH)N(R)₂,C(O)N(OR)R, and C(═NOR)R, wherein R can be hydrogen or a carbon-basedmoiety; for example, R can be hydrogen, (C₁-C₁₀₀)hydrocarbyl, alkyl,acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, orheteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or toadjacent nitrogen atoms can together with the nitrogen atom or atomsform a heterocyclyl.

The term “alkyl” as used herein refers to straight chain and branchedalkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from1 to 8 carbon atoms. Examples of straight chain alkyl groups includethose with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl,n-butyl, n-pentyl n-hexyl, n-heptyl, and n-octyl groups. Examples ofbranched alkyl groups include, but are not limited to, isopropyl,iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompassesn-alkyl, isoalkyl, and anteisoalkyl groups as well as other branchedchain forms of alkyl. Representative substituted alkyl groups can besubstituted one or more times with any of the groups listed herein, forexample, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, andhalogen groups.

The term “alkenyl” as used herein refers to straight and branched chainand cyclic alkyl groups as defined herein, except that at least onedouble bond exists between two carbon atoms. Thus, alkenyl groups havefrom 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12carbons or, in some embodiments, from 2 to 8 carbon atoms. Examplesinclude, but are not limited to vinyl, —CH═CH(CH₃), —CH═C(CH₃)₂,—C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, cyclohexenyl,cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienylamong others.

The term “alkynyl” as used herein refers to straight and branched chainalkyl groups, except that at least one triple bond exists between twocarbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 toabout 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments,from 2 to 8 carbon atoms. Examples include, but are not limited to—C≡CH, —C≡C(CH₃), —C≡C(CH₂CH₃), —CH₂C≡CH, —CH₂C≡C(CH₃), and—CH₂C≡C(CH₂CH₃) among others.

The term “acyl” as used herein refers to a group containing a carbonylmoiety wherein the group is bonded via the carbonyl carbon atom. Thecarbonyl carbon atom is bonded to a hydrogen forming a “formyl” group oris bonded to another carbon atom, which can be part of an alkyl, aryl,aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroarylalkyl group or the like. An acyl group can include0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atomsbonded to the carbonyl group. An acyl group can include double or triplebonds within the meaning herein. An acryloyl group is an example of anacyl group. An acyl group can also include heteroatoms within themeaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example ofan acyl group within the meaning herein. Other examples include acetyl,benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups andthe like. When the group containing the carbon atom that is bonded tothe carbonyl carbon atom contains a halogen, the group is termed a“haloacyl” group. An example is a trifluoroacetyl group.

The term “aryl” as used herein refers to cyclic aromatic hydrocarbonsthat do not contain heteroatoms in the ring. Thus aryl groups include,but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl,indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl,naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups.In some embodiments, aryl groups contain about 6 to about 14 carbons inthe ring portions of the groups. Aryl groups can be unsubstituted orsubstituted, as defined herein. Representative substituted aryl groupscan be mono-substituted or substituted more than once, such as, but notlimited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substitutednaphthyl groups, which can be substituted with carbon or non-carbongroups such as those listed herein.

The term “heterocyclyl” as used herein refers to aromatic andnon-aromatic ring compounds containing three or more ring members, ofwhich one or more is a heteroatom such as, but not limited to, N, O, andS.

The term “alkoxy” as used herein refers to an oxygen atom connected toan alkyl group, including a cycloalkyl group, as are defined herein.Examples of linear alkoxy groups include but are not limited to methoxy,ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples ofbranched alkoxy include but are not limited to isopropoxy, sec-butoxy,tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclicalkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can includeone to about 12-20 or about 12-40 carbon atoms bonded to the oxygenatom, and can further include double or triple bonds, and can alsoinclude heteroatoms. For example, an allyloxy group is an alkoxy groupwithin the meaning herein. A methoxyethoxy group is also an alkoxy groupwithin the meaning herein, as is a methylenedioxy group in a contextwhere two adjacent atoms of a structure are substituted therewith.

The term “amine” as used herein refers to primary, secondary, andtertiary amines having, e.g., the formula N(group)₃ wherein each groupcan independently be H or non-H, such as alkyl, aryl, and the like.Amines include but are not limited to R—NH₂, for example, alkylamines,arylamines, alkylarylamines; R₂NH wherein each R is independentlyselected, such as dialkylamines, diarylamines, aralkylamines,heterocyclylamines and the like; and R₃N wherein each R is independentlyselected, such as trialkylamines, dialkylarylamines, alkyldiarylamines,triarylamines, and the like. The term “amine” also includes ammoniumions as used herein.

The term “amino group” as used herein refers to a substituent of theform —NH₂, —NHR, —NR₂, —NR₃ ⁺, wherein each R is independently selected,and protonated forms of each, except for —NR₃ ⁺, which cannot beprotonated. Accordingly, any compound substituted with an amino groupcan be viewed as an amine. An “amino group” within the meaning hereincan be a primary, secondary, tertiary, or quaternary amino group. An“alkylamino” group includes a monoalkylamino, dialkylamino, andtrialkylamino group.

The terms “halo,” “halogen,” or “halide” group, as used herein, bythemselves or as part of another substituent, mean, unless otherwisestated, a fluorine, chlorine, bromine, or iodine atom.

The term “hydrocarbon” or “hydrocarbyl” as used herein refers to amolecule or functional group, respectively, that includes carbon andhydrogen atoms. The term can also refer to molecule or functional groupthat normally includes both carbon and hydrogen atoms but wherein allthe hydrogen atoms are substituted with other functional groups.

As used herein, the term “hydrocarbyl” refers to a functional groupderived from a straight chain, branched, or cyclic hydrocarbon, and canbe alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combinationthereof. Hydrocarbyl groups can be shown as (C_(a)-C_(b))hydrocarbyl,wherein a and b are integers and mean having any of a to b number ofcarbon atoms. For example, (C₁-C₄)hydrocarbyl means the hydrocarbylgroup can be methyl (C₁), ethyl (C₂), propyl (C₃), or butyl (C₄).(C₀-C_(b))hydrocarbyl means in certain embodiments there is nohydrocarbyl group.

The term “solvent” as used herein refers to a liquid that can dissolve asolid, another liquid, or a gas. Non-limiting examples of solvents aresilicones, organic compounds, water, alcohols, ionic liquids, andsupercritical fluids.

The term “number-average molecular weight” as used herein refers to theordinary arithmetic mean of the molecular weight of individual moleculesin a sample. It is defined as the total weight of all molecules in asample divided by the total number of molecules in the sample.Experimentally, the number-average molecular weight (M_(n)) isdetermined by analyzing a sample divided into molecular weight fractionsof species I having n_(i) molecules of molecular weight M_(i) throughthe formula M_(n)=ΣM_(i)n_(i)/Σn_(i). The number-average molecularweight can be measured by a variety of well-known methods including gelpermeation chromatography, spectroscopic end group analysis, andosmometry. If unspecified, molecular weights of polymers given hereinare number-average molecular weights.

The term “room temperature” as used herein refers to a temperature ofabout 15° C. to about 28° C.

As used herein, “degree of polymerization” is the number of repeatingunits in a polymer.

As used herein, the term “polymer” refers to a molecule having at leastone repeating unit and can include copolymers. As used herein, the term“oligomer” refers to a relatively low molecular weight polymer in whichthe number of repeating units can be, for example, from 1 to 10. Theproperties of the oligomer can vary with the removal of one or a few ofthe units.

The term “copolymer” as used herein refers to a polymer that includes atleast two different repeating units. A copolymer can include anysuitable number of repeating units.

The polymers described herein can terminate in any suitable way. In someembodiments, the polymers can terminate with an end group that isindependently chosen from a suitable polymerization initiator, —H, —OH,a substituted or unsubstituted (C₁-C₂₀)hydrocarbyl (e.g., (C₁-C₁₀)alkyl,(C₆-C₂₀)aryl, or an alkyne) interrupted with 0, 1, 2, or 3 groupsindependently selected from —O—, substituted or unsubstituted —NH—, and—S—, a poly(substituted or unsubstituted (C₁-C₂₀)hydrocarbyloxy), apoly(substituted or unsubstituted (C₁-C₂₀)hydrocarbylamino) and ahalogen.

In various embodiments, salts having a positively charged counterion caninclude any suitable positively charged counterion. For example, thecounterion can be ammonium(NH₄ ⁺), or an alkali metal such as sodium(Na⁺), potassium (K⁺), or lithium (Li⁺). In some embodiments, thecounterion can have a positive charge greater than +1, which can in someembodiments complex to multiple ionized groups, such as Zn²⁺, Al³⁺, oralkaline earth metals such as Ca²⁺ or Mg²⁺.

In various embodiments, salts having a negatively charged counterion caninclude any suitable negatively charged counterion. For example, thecounterion can be a halide, such as fluoride, chloride, iodide, orbromide. In other examples, the counterion can be nitrate, hydrogensulfate, dihydrogen phosphate, bicarbonate, nitrite, perchlorate,iodate, chlorate, bromate, chlorite, hypochlorite, hypobromite, cyanide,amide, cyanate, hydroxide, permanganate. The counterion can be aconjugate base of any carboxylic acid, such as acetate or formate. Insome embodiments, a counterion can have a negative charge greater than−1, which can in some embodiments complex to multiple ionized groups,such as oxide, sulfide, nitride, arsenate, phosphate, arsenite, hydrogenphosphate, sulfate, thiosulfate, sulfite, carbonate, chromate,dichromate, peroxide, or oxalate.

The p-phenylene ethynylene compounds described herein can includecounterions. For example, a p-phenylene ethynylene compound bearing a—N⁺(CH₃)₃ can have a negatively charged counterion, such as Br⁻ or I⁻,associated with it.

As used herein, the term “3-methylimidazolium” refers to a substituenthaving the structure

The wavy line indicates the point of attachment to the rest of themolecule.

As used herein, the term “(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-” refers to a substituenthaving the structure

The wavy line indicates the point of attachment to the rest of themolecule.

As used herein, a “cationic p-phenylene ethynylene compound” refers to ap-phenylene ethynylene compound that has a net positive charge.

As used herein, the term “anionic enzyme substrate” refers to an enzymesubstrate that has a net negative charge.

As used herein, an “anionic p-phenylene ethynylene compound” refers to ap-phenylene ethynylene compound that has a net negative charge.

As used herein, the term “biological sample” includes, withoutlimitation, cell cultures or extracts thereof, biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof and otherbiological fluids.

As used herein, the term “microbial spore” can refer to any suitablemicrobial spore, such as a eukaryotic spore or a bacterial spore.

As used herein, the term “anionic enzyme substrate” can refer to anysuitable anionic enzyme substrate that can be used as described herein.The anionic enzyme substrate can be a substrate that is sufficientlyhydrophobic but is not so large that it cannot form a complex with anoligomer. The anionic enzyme substrate can be a phospholipid. Theanionic enzyme substrate can be a lipopolysaccharide or other hybridspecies that can be a component of a membrane. The anionic enzymesubstrate can be an anionic peptide or a small protein with anet-negative patch. The anionic enzyme substrate can be DNA. The anionicenzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol) (DLPG).

As used herein, the term “cationic enzyme substrate” can refer to anysuitable cationic enzyme substrate that can be used as described herein.The cationic enzyme substrate can be a substrate that is not so largethat it cannot form a complex with an oligomer. The cationic enzymesubstrate can be a suitable cleavable amphiphilic substrate. Thecationic enzyme substrate can be a cationic phospholipid, such ascationic dimyristoyltrimethylammonium propane (DMTAP). The cationicenzyme substrate can be a cationic peptide or a small protein with anet-positive patch. The cationic enzyme substrate can be DNA. Thecationic enzyme substrate can be lauroyl choline. The cationic enzymesubstrate can be acetylcholine.

As used herein, the term “phenolate” refers to a p-phenolate, e.g., aphenolate attached via the 4-position.

p-Phenylene Ethynylene Compounds for Inducing Germination of MicrobialSpores.

In various embodiments, a method for inducing the germination ofmicrobial spores with a p-phenylene ethynylene compound is describedherein. The microbial spores can be at least one of a Bacillusanthracis, a Bacillus atrophaeus, a Bacillus cereus, and a Bacillussubtilis.

In various embodiments, the p-phenylene ethynylene compound includes arepeating unit which can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L² can be independently chosenfrom a (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 groups independently chosen from —O—, —S—, and —NH—. Thevariable R², at each occurrence, can be independently chosen from —H,(C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, phenolate,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, 3-methylimidazolium and—N⁺(R^(A))₃ wherein at each occurrence R^(A) is independently(C₁-C₅)alkyl. The variable L¹, at each occurrence can be independentlychosen from a bond and

The variable j can be about 0 to about 4.

In various embodiments, the p-phenylene ethynylene compound can includea repeating unit having the structure:

The variable R³, can be independently chosen from —N⁺(CH₃)₃,

The variable n can be about 2 to about 4.

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from a (C₁-C₅₀)hydrocarbylene interrupted by 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, 3-methylimidazolium andN⁺(R^(A))₃ wherein at each occurrence R^(A) is independently(C₁-C₅)alkyl. The variable j can be about 0 to about 4. The variable L¹,at each occurrence, can be independently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl,—C(O)NH—(C₁-C₁₀)hydrocarbyl, and C(O)OH. The variable m can be about 1to about 1,000.

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4. The variable A, at eachoccurrence can be independently chosen from —H and —C(O)O—(C₁-C₅)alkyl.In various embodiments, the variable A can be —C(O)OCH₂CH₃. The variableq can be about 1 to about 50, about 1 to about 40, about 1 to about 30,about 1 to about 20, about 1 to about 10, about 1 to about 5, about 1 toabout 3, and about 1, 2, 3, 4, 5, 7, 10, 20, 30, 40, and about 50 orgreater.

In various embodiments the p-phenylene ethynylene compound can have thestructure:

The variable R¹, at each occurrence can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can be a(C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 groups independently chosen from —O—, —S—, and —NH—. The variable R²,at each occurrence can be independently chosen from N⁺(R^(A))₃,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.The variable p can be about 1 to about 10, about 1 to about 7, about 1to about 5, about 1 to about 3, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, orabout 10 or greater.

In various embodiments, the variable Z, at each occurrence, can be —O—.The variable L², at each occurrence, can independently be a(C₁-C₅)alkyl. The variable R², at each occurrence, can be independentlychosen from —N⁺(CH³)₃,

The variable p can be about 1 to about 5, about 1 to about 3, or about1, 2, 3, 4, or about 5 or greater.

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable P can be about 1, 2, 3, 4, or about 5 or greater.

Method for Detecting Enzymes

In various embodiments, a method for detecting enzymes is describedherein. The method includes (i) introducing an enzyme to a compositionincluding a p-phenylene ethynylene compound and an enzyme substrate; and(ii) analyzing the change in fluorescence of the p-phenylene ethynylenecompound following introduction of the enzyme. The introducing step (i)can include introducing the p-phenylene ethynylene compound and theenzyme substrate to a sample which includes an enzyme. Further, theintroducing step (i) can include introducing a sample which includes anenzyme to the p-phenylene ethynylene compound and the enzyme substrate.In various embodiments, the p-phenylene ethynylene compound and theenzyme substrate form a complex. In various embodiments, theintroduction step (i) and the analyzing step (ii) occur in an aqueousenvironment.

In various embodiments, the fluorescence of the p-phenylene ethynylenecompound can decrease following the introduction of the enzyme. Thefluorescence can decrease due to a molecular transformation of theenzyme substrate to an entity or entities that do not complex with thep-phenylene ethynylene compound.

In various embodiments, the p-phenylene ethynylene compound can be acationic p-phenylene ethynylene compound. In various embodiments, thecationic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,phenolate, —N⁺(R^(A))₃, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.Further, at least one R² can be independently chosen from N⁺(R^(A))₃,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, suchthat the cationic p-phenylene ethynylene compound has a net positivecharge. The variable j can be about 0 to about 4, about 1 to about 2, or0, 1, 2, 3, or 4. The variable L¹, at each occurrence, can beindependently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4, or about 2, 3, or about 4 orgreater. The variable A, at each occurrence, is independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl. The variable q is about 1 to about 5,about 1 to about 3, or about 1, 2, 3, 4, or about 5 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be an anionic enzymesubstrate. For example, the anionic enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol) (DLPG), having thestructure:

In various embodiments, the p-phenylene ethynylene compound is ananionic p-phenylene ethynylene compound. In various embodiments, theanionic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,and phenolate. Further, at least one R² can be an anionic group such asSO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ⁻, or phenolate, such that theanionic p-phenylene ethynylene compound can have a net negative charge.The variable j can be about 0 to about 4, about 1 to about 2, or 0, 1,2, 3, or 4. The variable L¹, at each occurrence, can be independentlychosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen fromSO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate. The variable R³can be —SO₃ ⁻. The variable n can be about 2 to about 4, or about 2, 3,or about 4 or greater. The variable A, at each occurrence, isindependently chosen from —H and —C(O)O—(C₁-C₅)alkyl. The variable q isabout 1 to about 5, about 1 to about 3, or about 1, 2, 3, 4, or about 5or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3 or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be a cationic enzymesubstrate. The cationic enzyme substrate can be a substituted orunsubstituted (C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.The cationic enzyme substrate can be a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃. In various embodiments thecationic enzyme substrate can be chosen from lauroyl choline andacetylcholine. The cationic enzyme substrate can be lauroyl choline.

In various embodiments, the enzyme can be any suitable enzyme. Forexample, the enzyme can be a protolytic enzyme, a DNA restrictionenzyme, a phosphatase, or a kinase. In various embodiments, the enzymecan be chosen from phospholipase A1, phospholipase A2, phospholipase C,and acetyl cholinesterase.

In various embodiments, the method includes (i) introducing an enzyme toa composition including a p-phenylene ethynylene compound and an enzymesubstrate; and (ii) analyzing the change in fluorescence of thep-phenylene ethynylene compound following introduction of the enzyme.The p-phenylene ethynylene compound can have the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater. The enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol). In variousembodiments, the enzyme can be any suitable enzyme. For example, theenzyme can be a protolytic enzyme, a DNA restriction enzyme, aphosphatase, or a kinase. The enzyme can be at least one ofphospholipase A1, phospholipase A2, and phospholipase C.

In various embodiments, the method can include (i) introducing an enzymeto a composition including a p-phenylene ethynylene compound and anenzyme substrate; and (ii) analyzing the change in fluorescence of thep-phenylene ethynylene compound following introduction of the enzyme.The p-phenylene ethynylene compound can have the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater. The enzyme substrate can be lauroyl choline. The enzyme can beany suitable enzyme. The enzyme can be a phospholipase (e.g., PLA1,PLA2, PLC, PLB). The enzyme can be butyrylcholinesterase. The enzyme canbe acetylcholinesterase.

Sensor for Detecting Enzymes

In various embodiments a sensor is described herein. The sensor may be asensor for the detection of enzymes. The sensor includes a p-phenyleneethynylene compound and an enzyme substrate. In various embodiments, thep-phenylene ethynylene compound can be a charged p-phenylene ethynylenecompound and the enzyme substrate can be an oppositely charged enzymesubstrate. In various embodiments, the p-phenylene ethynylene compoundand the enzyme substrate form a complex.

In various embodiments, the p-phenylene ethynylene compound can be acationic p-phenylene ethynylene compound. In various embodiments, thecationic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,phenolate, —N⁺(R^(A))₃, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.Further, at least one R² can be independently chosen from N⁺(R^(A))₃,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, suchthat the cationic p-phenylene ethynylene compound has a net positivecharge. The variable j can be about 0 to about 4, about 1 to about 2, or0, 1, 2, 3, or 4. The variable L¹, at each occurrence, can beindependently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4, or about 2, 3, or about 4 orgreater. The variable A, at each occurrence, can be independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl. The variable q can be about 1 to about5, about 1 to about 3, or about 1, 2, 3, 4, or about 5 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be an anionic enzymesubstrate. For example, the anionic enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

In various embodiments, the p-phenylene ethynylene compound can be ananionic p-phenylene ethynylene compound. In various embodiments, theanionic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,and phenolate. Further, at least one R² can be an anionic substituentsuch as —SO₃ ⁻—, —CO₂ ⁻—, —H₂PO₄ ⁻—, HPO₄ ²⁻, PO₄ ³⁻, or phenolate, suchthat the anionic p-phenylene ethynylene compound can have a net negativecharge. The variable j can be about 0 to about 4, about 1 to about 2, or0, 1, 2, 3, or 4. The variable L¹, at each occurrence, can beindependently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate. The variableR³ can be —SO₃ ⁻. The variable n can be about 2 to about 4, or about 2,3, or about 4 or greater. The variable A, at each occurrence, can beindependently chosen from —H and —C(O)O—(C₁-C₅)alkyl. The variable q canbe about 1 to about 5, about 1 to about 3, or about 1, 2, 3, 4, or about5 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3 or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be a cationic enzymesubstrate. The cationic enzyme substrate can be a substituted orunsubstituted (C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.The cationic enzyme substrate can be a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃. In various embodiments thecationic enzyme substrate can be chosen from lauroyl choline andacetylcholine. The cationic enzyme substrate can be lauroyl choline.

In various embodiments the sensor includes a cationic p-phenyleneethynylene compound and an anionic enzyme substrate. The p-phenyleneethynylene compound can have the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater. The anionic enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

In various embodiments the sensor includes a p-phenylene ethynylenecompound and an enzyme substrate. The p-phenylene ethynylene compoundcan have the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater. The enzyme substrate can be lauroyl choline.

Method of Protein Analysis

In various embodiments, a method for protein analysis is describedherein. The method includes (i) introducing a p-phenylene ethynylenecompound to a biological sample including at least one protein and (ii)analyzing the fluorescence of the p-phenylene ethynylene compound in thepresence of the biological sample including the at least one protein.

It has been unexpectedly discovered that p-phenylene ethynylenecompounds can be employed for the selective detection of proteins inbiological samples. For example, p-phenylene ethynylene compounds can beused to detect the presence of amyloids in tissue samples. Further, ithas been unexpectedly discovered that p-phenylene ethynylene compoundsdisplay a distinguishable response to monomeric and fibrillary proteins.For example, p-phenylene ethynylene compounds show display adistinguishable response to monomeric and fibrillary Aβ-40 amyloid andα-synuclein.

In various embodiments, the fluorescence of the p-phenylene ethynylenecompound is analyzed prior to being introduced to the biological sampleincluding the at least one protein. In various embodiments, analyzingthe fluorescence of the p-phenylene ethynylene compound in the presenceof the biological sample including the at least one protein includesanalyzing the spectral changes between the fluorescence of thep-phenylene ethynylene compound of step (i) and the fluorescence of thep-phenylene ethynylene compound in the presence of the biological sampleincluding the at least one protein of step (ii). In various embodiments,the morphology of the protein in the biological sample is determined byanalyzing spectral changes between the fluorescence of the p-phenyleneethynylene compound of step (i) and the fluorescence of the p-phenyleneethynylene compound in the presence of the biological sample includingthe at least one protein of step (ii). In various embodiments, thespectral changes in the fluorescence of the p-phenylene ethynylenecompound between step (i) and step (ii) are induced by changes in theconformational freedom of the p-phenylene ethynylene compound betweenstep (i) and step (ii).

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,phenolate, —N⁺(R^(A))₃, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.The variable j can be about 0 to about 4, about 1 to about 2, or 0, 1,2, 3, or 4. The variable L¹, at each occurrence, can be independentlychosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4, or about 2, 3, or about 4 orgreater. The variable A, at each occurrence, can be independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl. The variable q can be about 1 to about5, about 1 to about 3, or about 1, 2, 3, 4, or about 5 or greater

In various embodiments, the p-phenylene ethynylene compound can have thestructure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater.

In various embodiments, the protein can be at least one of an amyloidbeta protein, Aβ-40, Aβ-42, tau, and α-synuclein, islet amyloidprecursor protein, Huntingtin, prion, lysozyme, TDP-43 (transactiveresponse DNA-binding protein 43), FUS (fused in sarcoma), and insulin.

In various embodiments, the method includes (i) analyzing thefluorescence of a p-phenylene ethynylene compound; (ii) introducing thep-phenylene ethynylene compound to a biological sample including atleast one protein; (iii) analyzing the fluorescence of the p-phenyleneethynylene compound in the presence of the biological sample includingthe at least one protein; and (iv) determining the morphology of the atleast one protein in the biological sample by analyzing spectral changesbetween the fluorescence of the p-phenylene ethynylene compound of step(i) and the fluorescence of the p-phenylene ethynylene compound in thepresence of the biological sample including the at least one protein ofstep (iii). The p-phenylene ethynylene compound can have the structure:

The variable s can be about 1 to about 3, such as about 1. The proteincan be at least one of an amyloid beta protein, Aβ-40, Aβ-42, tau, andα-synuclein, islet amyloid precursor protein, Huntingtin, prion,lysozyme, TDP-43 (transactive response DNA-binding protein 43), FUS(fused in sarcoma), and insulin.

Method of Detecting Chemical Agents

In various embodiments, a method for detecting a chemical agent isdescribed herein. The method includes (i) exposing a sensor compositionincluding a complex including a p-phenylene ethynylene compound and anenzyme substrate to a sample; (ii) introducing an enzyme to the sensorcomposition of step (i); and (iii) analyzing the change in fluorescenceof the p-phenylene ethynylene compound between the exposing step (i) andthe introducing an enzyme step (ii).

The sample may be, but is not limited to, any solution that has beenexposed to a potential chemical agent. Chemical agents include, withoutlimitation, organophosphate nerve agents (e.g. sarin, soman, tabun, VX,and VR) and G-type nerve agents (e.g. diethyl phosphoramidate). Thechemical agent can be a pesticide or insecticide, such as anorganophosphate pesticide or insecticide, such as malathion (e.g.,Diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate) orchlorpyrifos (e.g., O,O-diethyl O-3,5,6-trichloropyridin-2-ylphosphorothioate).

In various embodiments, a change in fluorescence between the exposingstep (i) and the introducing an enzyme step (ii) indicates the presenceof a chemical agent that does interact with the enzyme. In variousembodiments, a minimal change in fluorescence between the exposing step(i) and the introducing an enzyme step (ii) indicates the presence of achemical agent that does interact with the enzyme.

In various embodiments, the p-phenylene ethynylene compound can be acationic p-phenylene ethynylene compound. In various embodiments, thecationic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,phenolate, —N⁺(R^(A))₃, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.Further, at least one R² can be independently chosen from N⁺(R^(A))₃,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, suchthat the cationic p-phenylene ethynylene compound has a net positivecharge. The variable j can be about 0 to about 4, about 1 to about 2, or0, 1, 2, 3, or 4. The variable L¹, at each occurrence, can beindependently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4, or about 2, 3, or about 4 orgreater. The variable A, at each occurrence, can be independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl. The variable q can be about 1 to about5, about 1 to about 3, or about 1, 2, 3, 4, or about 5 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be an anionic enzymesubstrate. For example, the anionic enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol). In variousembodiments, the enzyme can be any suitable enzyme. For example, theenzyme can be a protolytic enzyme, a DNA restriction enzyme, aphosphatase, or a kinase. In various embodiments the enzyme is chosenfrom phospholipase A1, phospholipase A2 and phospholipase C.

In various embodiments, the p-phenylene ethynylene compound is ananionic p-phenylene ethynylene compound. In various embodiments, theanionic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,and phenolate. Further, at least one R² can be an anionic group such as—SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, or phenolate, such that theanionic p-phenylene ethynylene compound can have a net negative charge.The variable j can be about 0 to about 4, about 1 to about 2, or 0, 1,2, 3, or 4. The variable L¹, at each occurrence, can be independentlychosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can independently be chosen from—SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate. The variableR³ can be —SO₃ ⁻. The variable n can be about 2 to about 4, or about 2,3, or about 4 or greater. The variable A, at each occurrence, can beindependently chosen from —H and —C(O)O—(C₁-C₅)alkyl. The variable q canbe about 1 to about 5, about 1 to about 3, or about 1, 2, 3, 4, or about5 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3 or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be a cationic enzymesubstrate. The cationic enzyme substrate can be a substituted orunsubstituted (C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.The cationic enzyme substrate can be a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃. In various embodiments thecationic enzyme substrate can be chosen from lauroyl choline andacetylcholine. The cationic enzyme substrate can be lauroyl choline.

The enzyme can be any suitable enzyme. The enzyme can be a phospholipase(e.g., PLA1, PLA2, PLC, PLB). The enzyme can be butyrylcholinesterase.In various embodiments, the enzyme can be acetylcholinesterase.

In various embodiments includes (i) exposing a sensor compositionincluding a complex including a p-phenylene ethynylene compound and anenzyme substrate to a sample; (ii) introducing an enzyme to the sensorcomposition of step (i); and (iii) analyzing the change in fluorescenceof the p-phenylene ethynylene compound between the exposing step (i) andthe introducing an enzyme step (ii). The p-phenylene ethynylene compoundcan have the structure:

The variable s can be 1. The enzyme substrate can be lauroyl choline. Achange in fluorescence between the exposing step (i) and the introducingan enzyme step (ii) can indicate the presence of a chemical agent thatdoes interact with the enzyme.

Sensor for Detecting the Presence of a Chemical Agent

In various embodiments, a sensor for detecting the presence of achemical agent is described herein. The sensor including a sensorcomposition including a complex including a p-phenylene ethynylenecompound and an enzyme substrate.

In various embodiments, the the p-phenylene ethynylene compound can be acationic p-phenylene ethynylene compound. In various embodiments, thecationic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,phenolate, —N⁺(R^(A))₃, (C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium. Thevariable R^(A), at each occurrence, can be independently (C₁-C₅)alkyl.Further, at least one R² can be independently chosen from N⁺(R^(A))₃(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium suchthat the cationic p-phenylene ethynylene compound has a net positivecharge. The variable j can be about 0 to about 4, about 1 to about 2, or0, 1, 2, 3, or 4. The variable L¹, at each occurrence, can beindependently chosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—N⁺(CH₃)₃,

The variable n can be about 2 to about 4, or about 2, 3, or about 4 orgreater. The variable A, at each occurrence, can be independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl. The variable q can be about 1 to about5, about 1 to about 3, or about 1, 2, 3, 4, or about 5 or greater.

In various embodiments, the cationic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3, or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be an anionic enzymesubstrate. For example, the anionic enzyme substrate can be1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

In various embodiments, the p-phenylene ethynylene compound can be ananionic p-phenylene ethynylene compound. In various embodiments, theanionic p-phenylene ethynylene compound can have the structure:

The variable R¹ can have the structure:

The variable Z, at each occurrence, can be independently chosen from—CH₂—, —O—, —S—, and —NH—. The variable L², at each occurrence, can beindependently chosen from (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 groups independently chosen from —O—, —S—,and —NH—. The variable R², at each occurrence, can be independentlychosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,and phenolate. Further, at least one R² can be an anionic group such as—SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, or phenolate, such that theanionic p-phenylene ethynylene compound can have a net negative charge.The variable j can be about 0 to about 4, about 1 to about 2, or 0, 1,2, 3, or 4. The variable L¹, at each occurrence, can be independentlychosen from a bond and

The variable A, at each occurrence, can be independently chosen from —H,substituted or unsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl. The variable m can be about 1 to about 10,about 1 to about 7 about 1 to about 5, about 1 to about 3 or about 1, 2,3, 4, 5, 6, 7, 8, 9, or about 10 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable R³, at each occurrence, can be independently chosen from—SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate. The variableR³ can be —SO₃ ⁻. The variable n can be about 2 to about 4, or about 2,3, or about 4 or greater. The variable A, at each occurrence, can beindependently chosen from —H and —C(O)O—(C₁-C₅)alkyl. The variable q canbe about 1 to about 5, about 1 to about 3, or about 1, 2, 3, 4, or about5 or greater.

In various embodiments, the anionic p-phenylene ethynylene compound canhave the structure:

The variable s can be about 1 to about 3 or about 1, 2 or about 3 orgreater.

In various embodiments, the enzyme substrate can be a cationic enzymesubstrate. The cationic enzyme substrate can be a substituted orunsubstituted (C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.The cationic enzyme substrate can be a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃. In various embodiments thecationic enzyme substrate can be chosen from lauroyl choline andacetylcholine. The cationic enzyme substrate can be lauroyl choline.

In various embodiments, the sensor includes a sensor compositionincluding a complex including a p-phenylene ethynylene compound and anenzyme substrate. The p-phenylene ethynylene compound can have thestructure:

The variable s can be 1. The enzyme substrate can be lauroyl choline.

EXAMPLES

Various embodiments of the present invention can be better understood byreference to the following Examples which are offered by way ofillustration. The present invention is not limited to the Examples givenherein.

Example 1 Example 1.1 Growth and Preparation of Bacillus atrophaeusSpores

Bacillus atrophaeus (ATCC #9372) spores were obtained from 20%glycerol-suspended spore stock stored at −70° C. A working batch of B.atrophaeus spores was obtained by induced germination and subsequentsporulation on sporulation agar. Using a sterile inoculation loop, sporecolonies were scraped off the sporulation agar, suspended in sterile DIwater, and filtered through glass wool. Spores were then washed threetimes via centrifugation (15 minutes, 4.4 k RPM), with the pellet beingresuspended in 50% ethanol, and stored at 4° C. for 12 hours to providetime for vegetative cell death. Spores were then washed another threetimes via centrifugation and resuspension in sterile DI water. Sporeswere then aliquoted into glass vials and stored at 4° C. until use. Theconcentration of spores in these aliquots was determined with ahemocytometer.

Example 1.2 Growth and Preparation of Bacillus atrophaeus VegetativeCells

Bacillus atrophaeus vegetative cells were obtained by adding 1 mL ofprepared spore aliquot to 50 mL Bacto tryptic soy broth (TSB; BecktonDickinson), which was then incubated for 18 hours at 30° C. with shaking(250 RPM). Vegetative cells were prepared for analysis by a triple-washstep consisting of three centrifugations of 4.4 k RPM for 15 minuteseach; with pellets resuspended in 0.85% NaCl (physiological salinesolution). The resulting cell concentrations were determined with ahemocytometer.

Example 1.3 Bacillus atrophaeus Viability Testing

Viability testing was carried out at concentrations of 10⁷ bacteria/mLin 0.85% NaCl. p-Phenylene ethynylene compound (“PE”) concentrations of10 μg/mL and 20 μg/mL were used to target B. atrophaeus vegetative cellsand spores, respectively. Samples were prepared at volumes of 1 mL in0.5-dram glass vials (VWR; Radnor, Pa.), and exposed to light or darkconditions for varying time periods. Light experiments were carried outin a 10-lamp LZC-ORG photoreactor (Luzchem Research; Ontario, Canada)fitted with UVA lamps (Hitachi FL8BL-B) exhibiting a power density of0.975 mW/cm², over a wavelength range of 316-400 nm (centered at 350nm). Samples were loaded into a rotating carousel in the center of thephotoreactor to ensure uniform light exposure. Following light exposure,samples were stained with a membrane-permeable stain, SYTO 21 (LifeTechnologies; Carlsbad, Calif.), and a membrane-impermeable stain,propidium iodide (PI; Life Technologies). Membrane-permeable nucleicacid dyes such as SYTO 21 stain nucleic acids throughout a bacterium,independent of membrane damage. On the other hand, membrane-impermeablestains such as propidium iodide selectively stain nucleic acids inbacteria with compromised membranes. Vegetative cell samples werepermitted 15 minutes to stain at room temperature (in the dark), whilespores were allowed 45 minutes to stain at room temperature (also in thedark). Staining of spores with hydrophilic nucleic acid stains, such aspropidium iodide, takes longer than that of vegetative cells due to thefact that a spore's DNA is supercoiled within the inner membrane, whichis inherently impermeable to hydrophilic molecules.

Stained samples were then evaluated with an Accuri C6 (Becton Dickinson)flow cytometer equipped with a 488 nm, 50 mW laser. SYTO 21 fluorescencewas quantified with the FL1 detector at 530±15 nm; PI fluorescence wasquantified with the FL3 detector at wavelengths exceeding 670 nm.

Two thresholds were used in viability analysis; the first was a forwardscatter (FSC) threshold that ensured that only events exceeding 50,000FSC units were included in the data sets. Lowering the forward scatterthreshold to 50,000 helps ensure that small Bacillus spores aren'tomitted as events. The second threshold was specific to the FL1detector, and ensured that only events exhibiting some degree of SYTO 21uptake (at least 250 fluorescence units) were included as data points.Bacteria were analyzed at a nominal flow rate of 14 μL/min, with astream core diameter of 10 μm. All samples were evaluated until at least10,000 events had been recorded. Using B. atrophaeus vegetative cells,the live gate was based on untreated negative controls, while the deadgate was based on positive controls exposed to 70% ethanol for 60minutes. An additional gate was obtained to denote the fluorescenceregions of viable, untreated spores.

Example 1.4 SEM of Bacillus atrophaeus Vegetative Cells and Spores

Spores exposed to EO-PE (Th,C2), or simply 0.85% NaCl as a negativecontrol, were examined by scanning electron spectroscopy (SEM) (Quanta250 FEG SEM; FEI; Hillsboro, Oreg.). Samples were fixed in 2.5%glutaraldehyde overnight at room temperature, rinsed inphosphate-buffered saline (PBS), and subsequently dehydrated in ethanol.Dehydrated samples were sputercoated in approximately 12 nm of gold andpalladium under vacuum and subsequently analyzed by SEM.

Example 1.5 Growth and Preparation of Bacillus anthracis Sterne

BSL2 agent B. anthracis Sterne was not permitted for use in flowcytometry facilities and thus was prepared and evaluated differentlyfrom the aforementioned B. atrophaeus. B. anthracis Sterne spore stockswere prepared as previously described. Briefly, spores were prepared inphage assay broth; sporulation was subsequently confirmed withphase-contrast microscopy, and any remaining vegetative cells werekilled with a 40 minute, 68° C. heat treatment. Bacteria were thenwashed and resuspended in Dulbecco's phosphate-buffered saline (DPBS;Gibco), tittered, aliquoted, and stored at −80° C. Colony growth ofaliquots was evaluated before and after heat treatment (40 minutes at68° C.) to ensure the absence of vegetative cells.

B. anthracis Sterne vegetative cells were prepared exclusively for theexperiments described herein. B. anthracis Sterne spores were removedfrom frozen storage and thawed at room temperature before 20 μL ofsuspension was streaked onto a tryptic soy agar (TSA) plate. TSA plateswere incubated for 16 hours at 37° C. A sterile inoculation loop wasthen used to transfer 2 colonies from the TSA plate into 40 mL of brainheart infusion (BHI), along with 200 μL of glycerol. The flask was thenaerobically incubated for 16 hours at 37° C. with 250 RPM shaking. Toensure sterility of BHI a second flask was aerobically incubated with 10mL BHI. Following verification of sterility, 200 μL of the inoculatedBHI was added to 800 μL sterile BHI, vortexed, and subsequentlytransferred to a disposable cuvette. The absorbance of the cuvette wasmeasured at 600 nm relative to sterile BHI. A subculture was thenprepared at an OD of 0.1 and aerobically incubated at 37° C. withshaking until the subculture's OD reached 1.0—indicating a vegetativecell concentration of 2×10⁷ CFU/mL (confirmed by colony growth on TSA).

Saline-washed B. anthracis Sterne vegetative cells were exposed to 10μg/mL EO-PE (Th,C2) in light and dark conditions for varying timedurations, diluted, and streaked onto TSA plates. TSA plates wereincubated at 37° C. for 18 hours and the colonies counted to estimateviability. B. anthracis Sterne spores were evaluated by a similartechnique, albeit with the implementation of heat treatment. In thiscase, samples were diluted, plated, and subsequently heat-treated tokill vegetative cells. Heat treatment is applied via a 68° C. water bathfor 30 minutes, such that B. atrophaeus vegetative cells are killed;presumably, any resulting colony growth would result from spores and notvegetative cells. Heat-treated samples were also diluted and plated—thedifference of colony growth between the heat-treated and non-heattreated samples was used to gauge the percentage of sample which wasvegetative cells. Negative controls contained no PE, while heattreatment consisted of submersion in a 68° C. water bath for 30 minutes.Equations 1.1, 1.2, and 1.3 were used to infer sample populations basedon colony growth:

$\begin{matrix}{{\% \mspace{14mu} {of}\mspace{14mu} {CFUs}\mspace{14mu} {that}\mspace{20mu} {are}\mspace{14mu} {Viable}\mspace{14mu} {Vegetative}\mspace{14mu} {Cells}} = {100 \times \frac{{CFU} - {CFU}_{HT}}{CFU}}} & (1.1) \\{{\% \mspace{14mu} {of}\mspace{14mu} {CFUs}\mspace{14mu} {that}\mspace{14mu} {are}\mspace{14mu} {Viable}\mspace{14mu} {Spores}} = {100 \times \frac{{CFU}_{HT}}{CFU}}} & (1.2) \\{\% \mspace{14mu} {of}\mspace{14mu} {CFUs}\mspace{14mu} {that}\mspace{14mu} {are}\mspace{14mu} {nonviable}\mspace{14mu} {relativeve}\mspace{14mu} {to}\mspace{14mu} {negative}\mspace{14mu} {control}\mspace{14mu} 100 \times \frac{{CFU}_{NC} - {CFU}}{{CFU}_{NC}}} & (1.3)\end{matrix}$

As used herein, “CFUs” and “CFU” refer to the number of colony formingunits on TSA prior to Heat Treatment, CFU_(HT) is the number of colonyforming units on TSA after Heat Treatment, and CFU_(NC) is the number ofcolony forming units on TSA in the negative control (no heat treatment).These equations are implemented in FIG. 5 as a means to monitorviability and germination, under the assumption that heat treatedvegetative cells lose their ability to form colonies. All B. anthracisSterne experiments (those shown in FIGS. 4 and 5) were conducted intriplicate.

Example 1.5 Results

PE-induced cell death was inferred using a complementary set of nucleicacid stains such that bacteria with intact cell membranes exhibit uptakeof SYTO 21, while only cells with compromised membranes exhibit uptakeof PI. In these studies, flow cytometry was utilized to gauge cellviability by rapid interrogation of dual-stain fluorescence. FIGS. 1A-Dillustrate flow cytometry-reported viability of 10,000 B. atrophaeusvegetative cells determined by changes in PI (X-axis) and SYTO 21(Y-axis) fluorescence; the L Gate represents the live vegetative cellsand the D gate represents the dead vegetative cells; B. atrophaeusvegetative were cells suspended in physiological saline solution for 1hr; A: negative control (0 μg/mL PE); B: 10 μg/mL PE in the dark; C:negative control (0 μg/mL) in UVA light; D: 10 μg/mL PE in UVA light.

FIG. 1A illustrates fluorescence emitted from untreated B. atrophaeusvegetative cells: 1 hr in physiological saline solution at a temperatureof 28.5° C. Under these conditions, 100% of untreated B. atrophaeusvegetative cells retained their ability to form colonies and exhibitgreater uptake of SYTO 21 (˜10⁵ arbitrary fluorescence units) than PI(−10⁴ arbitrary fluorescence units). In this case, cellular membranesremained intact, limiting propidium iodide uptake. The addition of aknown membrane-disrupting agent, such as EO-PE (Th,C2), results in anoticeable fluorescence shift (FIG. 1B). The structure of EO-PE (Th,C2)is depicted below in Scheme 1. Following an hour's exposure to this PEat a concentration of 10 μg/mL, B. atrophaeus cells exhibit uptake ofSYTO 21 with the same propensity as untreated cells (FIG. 1A); however,the uptake of PI has increased tenfold, demonstrating that PEs inducemoderate membrane damage to the extent that 73% of cells are killed inthe absence of light. FIG. 1C shows that B. atrophaeus vegetative cellsare somewhat vulnerable to UVA irradiation, with 3% of bacilli killed inan hour. Significant killing of B. atrophaeus vegetative cells was onlyachieved upon exposure to light-activated PE; an exposure duration of 1hour causes 99% cell death (FIG. 1D). It is important to note thatmembrane damage to vegetative bacilli rarely results in non-specificuptake of both stains; that is, the uptake of one stain appears to occurindependently of the other.

Having defined regions of fluorescence characteristic of viable andnon-viable B. atrophaeus vegetative cells, flow cytometry was thenutilized to determine the extent by which EO-PE (Th,C2) inflicts damageon B. atrophaeus spores. Spores were treated under the same conditionsas the aforementioned vegetative cells (depicted in FIGS. 1A-D);however, spores were stained with SYTO 21 and PI for 45 minutes, whereasvegetative cells needed just 15 minutes to stain.

FIGS. 2A-D illustrate flow cytometry-reported germination of 10,000 B.atrophaeus spores determined by changes in PI (X-axis) and SYTO 21(Y-axis) fluorescence; the S gate represents spores, the L Gaterepresents viable germinating spores, and the D gate represents cellsthat have died during germination; B. atrophaeus spores were suspendedin physiological saline solution for 1 hr; A: negative control (0 μg/mLPE) in the dark; B: 20 μg/mL PE in the dark; C: negative control (0μg/mL PE) in UVA light; D: 20 μg/mL PE in UVA light. Despite a prolongedstaining period, the untreated B. atrophaeus spores depicted in FIG. 2Aexhibit decreased uptake of both stains relative to the untreatedvegetative cells shown in FIG. 1A. Decreased nucleic acid staining inBacillus spores is attributed to their low water content, relativelysmall volume, and limited access of stains to centralized supercoiledDNA resulting from an intact inner spore membrane. The S gate istherefore included in FIGS. 2A-D to indicate regions of fluorescencecharacteristic of untreated B. atrophaeus spores. As can be seen in FIG.2B, the addition of EO-PE (Th,C2) affects spores' stain uptake in adifferent manner than was observed with B. atrophaeus vegetative cellsin FIG. 1B. While exposure to PEs in the dark selectively enhanceduptake of PI in vegetative cells, the uptake of SYTO 21 is alsosignificantly enhanced in spores following PE exposure in the dark. Thisnon-specific fluorescent enhancement is characteristic of Bacillus sporegermination: within 10 minutes of induced germination, spore coatporosity rapidly increases, leading to a water influx event, and thus,increased cell volume and increased uptake of stains.

This method of interrogation suggests that the water influx eventsufficiently progressed in 39% of spores to the point where themagnitude of stain uptake matched that of B. atrophaeus vegetativecells. FIG. 2B shows that 61% of spores still fluoresce in a mannercharacteristic of that of untreated spores (the S gate), which suggeststhese spores either underwent delayed germination or were not induced togerminate at all. In the absence of light, PE-exposed spores were foundto germinate into viable vegetative cells—meaning they were capable offorming colonies on TSA, while also exhibiting fluorescencecharacteristic of late-log-phase vegetative cells.

Irradiating B. atrophaeus spores with UVA light, alone, does not affectstain uptake, as is shown in FIG. 2C. FIG. 2D shows that exposing sporesto light-activated PE also results in non-specific stain uptake;however, in this case, the uptake ratio of membrane impermeable stain(PI) to membrane permeable stain (SYTO 21) is increased, resulting infailed germination and inability to form colonies. Therefore,light-activated PE promptly kills most germinating spores—notsurprising, given the susceptibility of Bacillus vegetative cells tolight-activated PE (FIG. 1D). Increasing the PE concentration to 50 or100 μg/mL was found to have no increased effect on the death orgermination of B. atrophaeus spores, presumably to the inner filtereffect.

The presence of UVA light is significant in achieving significantbiocidal activity with cationic PEs, as light-induced inactivation of B.anthracis Sterne is believed to involve three steps. First, the PE isexcited from the ground state, S₀, to its excited singlet state, S₁.Second, via intersystem crossing, S₁ decays to a longer-lived, albeitlower energy, triplet state, S₃; in turn, S₃ transfers its energy tomolecular triplet oxygen, subsequently generating singlet oxygen (¹O₂)and ROS via a type I and type II photoreactions, respectively. Thirdly,ROS and ¹O₂ can locally oxidize lipids, proteins, and nucleic acids. Itis evident that the additional level of PE-inflicted damage conferred bythe presence of UVA light plays a major role in viability of germinatingB. atrophaeus spores. The additional damage inflicted by lightactivation of PE (as opposed to PE in the dark) may stunt germination bylimiting the progression of one or both water influx events, as bothfluorescence magnitudes (FIG. 2D) and forward scatter values aremitigated in this scenario.

The ability of PEs to induce germination of B. atrophaeus spores wasconfirmed with scanning electron microscopy (SEM). FIGS. 3A-E illustratescanning electron microscope images of B. atrophaeus spores (withvegetative cells that arose from PE-induced spore germination alsovisible); A: spores suspended in physiological saline solution for 5 hrsin the dark; B: spores exposed to 20 μg/mL PE for 5 hrs in the dark; C:spores suspended in physiological saline solution for 5 hrs in UVAlight; D and E: spores exposed to 20 μg/mL PE for 5 hrs in UVA light.Scale bars spanning 3 μm are included. Arrows indicate spore coatremnants. As shown in FIGS. 3A-E, SEM imaging illustrates an increasedquantity of germinating cells following their exposure to PE in thedark, as evidenced by their increased volume, rod-like morphology, andspore coat remnants. Vegetative cells were present in the negativecontrol, although they were far outnumbered by spores (the gating schemeused in FIG. 2A indicates that spores represent 3% of all bacteria, inthis instance). The observed dimensions of the rod-like vegetative cells(diameter: 0.8 μm; length: 2-3 μm) and spores (width: 0.7 μm; length:1.8 μm) both match previously reported records. Arrows are included(FIGS. 3B and 3D) to highlight the presence of spore coat remnants—thepresence of which generally coincides with one of the last stages ofspore germination. In the case of many bacilli, however, there is nosign of a spore coat remnant, signifying that there is a large degree ofdisparity concerning germination progress across the samplepopulation—corroborating the heterogeneous fluorescence exhibited bygerminating B. atrophaeus spores seen via flow cytometry. Whilegermination is still observed in the presence of light-activated PE, themajority of cells exhibit significant morphological damage that is toosevere for the bacterium to overcome (FIGS. 3D and 3E).

In order to accurately determine the viability of Bacillus spores andsubsequently germinated vegetative cells with accuracy, Bacillusanthracis Sterne was evaluated by standard plating techniques. FIG. 4illustrates B. anthracis Sterne vegetative cell viability followingexposure to 10 μg/mL PE. NC denotes Negative Controls, where PE was notused. FIG. 5 illustrates B. anthracis Sterne spore and germinatedvegetative cell viability as a function of starting concentration. PEexposed to varying spore concentrations for 90-min durations in theabsence (A) and presence (B) of UVA light. Viability was inferred basedon the spore's capacity to grow colonies on TSA before and after heattreatment, as described by Equations 1.1-1.3. FIG. 4 illustrates theprofound killing of B. anthracis Sterne vegetative cells in the presenceof light-activated PE, thereby corroborating the rapid death ofgerminating spores observed in FIG. 5. Once again, standard platingtechniques are implemented, with colony growth being used as thedetermining factor in viability. Nearly 4 log reduction of B. anthracisSterne vegetative cells is observed within 30 minutes; within 90minutes, 5 log reduction is observed.

FIG. 5 illustrates that, within 90 minutes, 81% of spores were inducedto germinate, even though the large majority (72% of all spores) are notkilled and thus achieve successful germination into a viable,colony-forming vegetative cell. The percentage of spores able togerminate into viable vegetative cells actually decreased when thestarting concentration of spores was lessened. Germination still occursin the presence of UVA light; however, the resulting viability ofgerminated vegetative cells is severely compromised in this case. 90minutes is a sufficient amount of time for light-activated PEs to inducegermination in B. anthracis Sterne spores and subsequently damageresulting vegetative cells to the point where they are incapable ofcolony growth. Exposing B. anthracis Sterne spores at a relatively highPE-to-spore ratio (20 ng PE/spore) resulted in 99% killing within just90 minutes.

FCS Express (De Novo Software) was used to quantify the fraction of allspores that germinate upon exposure to UVA light and/or oligomer,according to the florescence gates defined in FIGS. 1A-D and 2A-D. Eventhough exposure of B. atrophaeus spores to PE in the dark facilitatesgermination within 30 minutes, longer exposure times only marginallyincreased the percentage of spores that are induced to germinate.Increasing the exposure time beyond 90 minutes did not increase thepercentage of spores that were induced to germinate—presumably due to PEphotodegradation and loss of biocidal efficacy. In the presence oflight-activated PE, just 15% of spores will successfully germinate.Furthermore, in the case of both B. atrophaeus and B. anthracis Sternespores, 20 μg/mL PE was insufficient to induce complete germination. Inan effort to induce complete spore germination, the startingconcentration of spores was diminished, with the PE concentration heldconstant at 20 μg/mL.

Example 2 Example 2.1 Materials

−1 C was synthesized as previously described. +2C was synthesized aspreviously reported. Both PEs are light yellow solids, and readilydissolve in aqueous solution. Lauroyl choline chloride (Tokyo ChemicalIndustry Co.; Tokyo, Japan) was obtained as a solid powder and thecontainer was stored under vacuum over dessicant.1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG) (AvantiPolar Lipids, Alabaster, Ala.) was obtained as a lyophilized solidpowder, and was dissolved in methanol and stored at −21° C. prior touse. Phospholipase A₁ (PLA1) from Thermomyces lanuginosus was obtained(Sigma-Aldrich, St. Louis, Mo.) as a liquid solution with aconcentration of 10,000 Units/g. Phospholipase A₂ (PLA2) from Crotalusadamanteus venom was obtained as a lyophilized power with buffer saltsat an activity of 320 U/mg (Worthington Biochemical, Lakewood, N.J.).Acetylcholinesterase (AChE) from human erythrocytes was obtained as a pH8.0 buffered solution with an activity of >500 U/mg (Sigma-Aldrich, St.Louis, Mo.). A “unit (U) of PLA2 activity,” as referred to herein, ismeasured as the amount of enzyme needed to release one micromole oftitratable fatty acid per minute at pH 8.9 and 25° C. from lecithinemulsion. A “unit of activity for PLA1,” as referred to herein, isdefined the same, except at a pH of 7.5 AChE activity units are definedsimilarly, with one micromole of acetylthiocholine iodide hydrolyzed perminute at pH 7.4 and 37° C. The AChE inhibitors Meptazinol HCl(3-(3-ethylhexahydro-1-methyl-1H-azepin-3-yl)-phenol hydrochloride),Itopride HCl(N-[[4-[2-(Dimethylamino)ethoxy]phenyl]methyl]-3,4-dimethoxybenzamidehydrochloride), and TAE-1(2,2′,2′-[1,3,5-Triazine-2,4,6-triyltris(oxy-4,1-phenylenecarbonyloxy)]tris[N,N,N-trimethyl-ethanaminiumtri-iodide) were obtained as solids (Sigma-Aldrich, St. Louis, Mo.). Allsolutions were prepared using filtered water with a resistivity of >18.2MΩ*cm (EMD Millipore, Billerica, Mass.), with a pH of 7.5. The PEstested were as shown below in Scheme 2.

Example 2.2 Sample Preparation

A typical preparation of a −1C/lauroyl choline(“LaCh”) sensor is given.In a quartz cuvette with stirring, 20 μL of 500 mM−1C is added to 1970μL of water. After 15-30 seconds of mixing, 10 μL of a 2 mg/mL solutionof LaCh is added and allowed to mix for several minutes. A similarprocedure is followed for the preparation of the +2C/DLPG sensor.Typical sensors used in experiments with +2C/DLPG had concentrations of1.4 μM PE and DLPG concentrations of 16 μM. For the AChE sensor, 5 μM of−1C was used, and LaCh used for enzyme studies was 32 μM. Enzymeconcentrations in the range of 50 to 0.5 mU were tested for PLA1 andPLA2, and the range for AChE was 0.1 to 0.8 U.

Example 2.3 Absorbance and Fluorescence Spectroscopy

UV-visible absorption spectra were obtained using a Lambda-35 UV-VISSpectrometer fitted with a temperature-controlled cell with magneticstirring (Perkin Elmer, Waltham, Mass.). Fluorescence spectra wereobtained using a Photon Technology International fluorescencespectrometer equipped with a 75 W xenon arc lamp housed in an ellipticalreflector (Photon Technology International Birmingham, N.J.).Fluorescence quantum yields were calculated using the comparative methodrelative to the previously reported value for +2C. Least-squares linearregressions for substrate concentration calibration and fluorescencequantum yield determinations were performed using the software Origin 9.

Example 2.4 Detection of Enzyme Activity

The monitoring of the sensor was performed using the absorbancewavelength of 430 nm and the fluorescence wavelength of 440 nm(excitation of 370 nm for −1C, 375 nm for +2C). For both absorbance andfluorescence measurements of the sensor upon addition of enzyme, thesensor was prepared as described above in a quartz cuvette with constantstirring at room temperature (25° C.). The lid of the instrument wasquickly lifted and enzyme injected, resulting in a ˜0.5 second delay inthe initial change registered. Enzyme kinetics were determined for PLA1and PLA2 by converting the intensity of fluorescence or absorbance ofthe aggregate to substrate (DLPG) concentration, as given in equation(2.1), below.

$\begin{matrix}{\lbrack S\rbrack_{t} = {\lbrack S\rbrack_{0}\frac{\left( \frac{I_{t}}{I_{b}} \right) - 1}{\left( \frac{I_{0}}{I_{b}} \right) - 1}}} & (2.1)\end{matrix}$

Where: [S]_(t) denotes substrate concentration at time t, [S]₀ isinitial substrate concentration, I_(t) is fluorescence intensity at timet, I₀ is initial fluorescence intensity, I_(b) is backgroundfluorescence intensity. Once the fluorescence at 440 nm or absorbance at430 nm is converted to substrate concentration, standardMichaelis-Menten kinetics can be used. Non-linear fitting to a velocityvs substrate concentration plot was performed using the Hill equationusing Origin 9, with the formula given in equation (2.2). TheMichaelis-Menten equation serves as a special example of the Hillequation, and when n=1 the Hill equation is equivalent to theMichaelis-Menten equation commonly used for enzyme kinetics.

$\begin{matrix}{y = \frac{V_{\max} \cdot x^{n}}{\left( {k_{m}^{n} + x^{n}} \right)}} & (2.2)\end{matrix}$

Where n=cooperativity, V_(max) is the max velocity in μMol/min*mg orμM/min, and k_(m), which is the substrate concentration at half ofV_(max).

Example 2.5 Computational Methods

PEs were parametrized to the generalized Amber forcefield (GAFF)framework using the antechamber program in AmberTools12. The Lipid14parameters for Amber were used for the lipid1,2-dioleoyl-sn-glycero-3-phospho-1′-rac-glycerol (DOPG). The Gaussian09software package was used for all quantum-level calculations for residueparametrization, with geometry optimized at the B3LYP/6-31g** level andelectrostatic potentials used for residue parametrization derived withHartree-Fock and a 6-31g** basis set. GAFF atomtypes were used to assignVan der Waals parameters and bonding force constants. The assignedpartial charges of the PE from the quantum-level calculations werefitted using the RESP charge fitting method. The initial systemconfigurations were prepared using the program Packmol. Systems weresolvated with water and neutralized with sodium and chloride ions, andthe TIP3 water model was used. Simulations used full PME electrostaticsand cubic periodic boundary conditions. The system was first minimizedusing the steepest descent method for 2500 steps, followed by a 250 stepgradient minimization. Heating was carried out from 0 K to 100 K in 500ps, and then from 100 K to 303.15 K in 500 ps using the NVT ensemble.Simulations were performed for 100-250 ns using the NPT ensemble withthe Langevin barostat and thermostat with a time constant of 1/ps. TheAmber12-GPU software package was used with SPFP precision. Radialdistribution functions were measured over the simulation trajectoryusing the center of masses of the individual PEs using the cpptrajprogram in AmberTools. In order to sort out the most likely aggregatedform of an PE dimer, cpptraj was used to cluster interacting pairs ofPEs with the hierarchical agglomerative approach. The distance betweenframes was calculated using best-fit RMSD of the coordinates, andclustering analysis was carried out for PEs within 5 Å apart. UCSFChimera version 1.10 was used for rendering snapshots of thetrajectories and further clustering of the trajectories of the topclustered results from cpptraj, based on pairwise best-fitroot-mean-square deviations between separate PEs, to distinguish commonaggregate structures and provide a graphical representation of theclusters over time.

Example 2.6 Photophysical Effects of Complex Formation

The fluorescence detection of enzyme activity on lipids or lauroylcholine was enabled by the strong photophysical changes which occurredupon aggregation of the PEs. The changes in absorbance and fluorescencespectra of +2C with DLPG and −1C with LaCh are shown in FIGS. 6A-D.FIGS. 6A-D illustrate absorbance and fluorescence Spectra of (A)absorbance and (B) fluorescence (Ex: 375 nm) of 1.4 μM+2C with DLPG; (C)absorbance and (D) fluorescence (Ex: 370 nm) of 5 μM−1C with LaCh; allspectra indicate the varying DLPG/LaCh concentration or ratio ofsubstrate to PE.

As can be seen in FIGS. 6A-D, there are significant changes in thefluorescence and absorbance of aggregates of both anionic and cationicPEs. Interestingly, the changes resulting from aggregation are verysimilar for the +2C/DLPG and −1C/LaCh complexes. The absorbance spectrumis strongly red-shifted, with the major transition moving from 375 to440 nm. The minor band at ˜320 nm forms a bimodal shape with a secondpeak at 330 nm for −1C and 340 nm for +2C upon aggregation. In asolution of 0.5 OD or higher, the aggregates give a transparent yellowsolution with a slight bluish haze. In addition to the strong changes toabsorbance, the fluorescence is significantly altered upon aggregationwith the substrate molecules. The most significant effect which can beutilized for sensing is a strong enhancement of fluorescence from abroadened, weak fluorescence to a very strong, structured emissioncentered at 450 nm for +2C and 442 nm for −1C.

The spectra in FIGS. 6A-D also show that the aggregates result instructured bands in both the absorbance and fluorescence spectra whichare within 10 nm apart. This suggests that a very highly-efficientfluorescence is occurring, resulting in very little energy loss and avery active sensor. This contrasts significantly with thenon-fluorescent, uncolored compound before complexation. The red-shiftedabsorbance and enhanced fluorescence is typical of a “J-aggregate”,which leads to the prediction that the molecular structure that resultsin these enhanced electronic properties allows these rigid molecules toalign. Comparing FIG. 6B with FIG. 6D, it is clear that the −1C/LaChaggregate has a more dominant structured band at ˜440 nm than the+2C/DLPG aggregate. This suggests that the structure of the −1C/LaChaggregate is that of a well-defined J-dimer, where the +2C/DLPGaggregate likely is also a J-dimer but with more conformational freedom.This result of fluorescence enhancement suggests that the fluorescencequantum yields would be useful for describing the enhancement by theaggregation.

FIGS. 7A-B illustrates integrated fluorescence versus absorbance for (A)5 uM−1C (squares) and 5 uM−1C with 32 uM LaCh (diamonds), and (B) 1.4uM+2C (squares) and 1.4 uM+2C with 16 uM DLPG (diamonds). This data wasused to calculate the fluorescence quantum yields by the comparativemethod, and the new values for the quantum yields that were correctedfrom a previous study are given next to the line. Excitation was 370 nmfor A and 375 nm for B, with fluorescence excitation wavelengths from390 to 600 nm. Fluorescence quantum yields were determined using thecomparative method as discussed above, and the least-squares linearregressions of the results are given in FIGS. 7A-B. FIGS. 7A-Bdemonstrate that the quantum yields of the PEs are greatly enhanced bythe aggregation induced by the substrates DLPG or LaCh. Calculation ofthe fluorescence yield by the comparative method using the reportedvalue of 0.039 for +2C leads to vastly overstated quantum yields offluorescence of both AChE and PLA sensors in excess of unity. While itis understandable that the previous value of +2C was difficult topinpoint due to the very low fluorescence of +2C in water, the resultsof fluorescence quantum yield measurements performed in this studysuggest that the quantum yield for +2C is no larger than 0.016 ratherthan 0.039. This value would assume a quantum yield for the −1C/LaChcomplex of near unity, and while the aggregation-induced fluorescence isextremely efficient, the quantum yield is more likely between 90 and 100percent. The aggregation of +2C with DLPG results in a fluorescenceenhancement 39 times at 440 nm, which correlates to an increase offluorescence quantum yield from 0.016 to 0.63. The aggregation of −1Cwith LaCh results in a considerable enhancement of fluorescence quantumyield, which is 66 times higher for the −1C/LaCh complex than −1C alone.This correlates with an enhancement from 0.015 to 0.991, following thecorrection of the quantum yield of fluorescence of +2C from 0.039 to0.016.

In addition to the strong changes in absorbance and fluorescence of thePEs, the formation of a complex can be confirmed through circulardichroism (CD) spectroscopy. Circular dichroism spectra of 1.4 uM+2Cwith and without 16 uM DLPG added were acquired, and illustrated that+2C strongly absorbs circularly polarized light with a strong negativeband at 445 nm. Since DLPG is chiral, it is reasonable that an aggregateformed on a DLPG template would be optically active. The photophysicalchanges observed upon complexation allow for a variety of strategies forindication of the presence of a substrate. While the magnitude of thefluorescence enhancement is much greater than that of the absorbancechange, the ability to use colorimetric means for determinations allowsfor cheaper and more flexible detection strategies than are accessibleby fluorescence measurements. The aggregation with surfactants andsubstrates is useful, and the introduction of substrates which aredegradable by enzymes allows use of PEs as fluorescence-quenching enzymesensors. The use of PEs for sensing of enzyme activity is powerful, astheir strong fluorescence quenching and dequenching allows for highlysensitive detection.

The aggregate formed between −1C and lauroyl choline gives rise toevidence of a structured aggregate with “J-type” character. In order tofurther investigate the structure of the aggregate that is formed, a setof large-scale molecular simulations was carried out.

Example 2.7 Molecular Aggregates for Monitoring EnzymeActivity—Phospholipases A1, A2 and C

The use of lipids such as DLPG to induce aggregation allows for thecreation of a sensor which can be affected by phospholipases.Phospholipases are a class of phosphodiesterases that can cleave theacyl chains or phosphate groups of the lipids, depending on the class.Phospholipases A1 and A2 (“PLA1,” “PLA2”) cleave the SN-1 and SN-2 acylchains respectively, while Phospholipase C (“PLC”) cleaves before thephosphate, forming diacylglycerol and a phosphate-containing headgroup.Observation of the effects of these three different enzymes on thephotophysical properties of the +2C/DLPG complex allow for assessment ofthe ability of the enzymatic products to maintain aggregation of thePEs. The changes in absorbance and fluorescence of the +2C/DLPGcomplexes were monitored after addition of either PLA1, PLA2, or PLC tothe solution as described in the methods section. In addition to varyingenzyme concentration, a study varying the concentration of DLPG was alsocarried out to determine changes in the response rate of the sensor whenexcess lipid is present.

The effects of enzymatic activity on the +2C/DLPG sensor are shown forPLA1 and PLA2 in FIGS. 8A-D. FIGS. 8A-D illustrate fluorescence versustime for PE with various concentrations of DLPG and PLA1 or PLA2; (A)fluorescence of the +2C/DLPG aggregates over the course of PLA1 activitywith 1.4 μM PE and a DLPG concentration of 7.27 μM, with enzyme addedranging from 0.5 to 5 mU of PLA1; (B) 1.4 μM of +2C with DLPG at aseries of concentrations from 10.6 to 35.6 μM (7.5-25.4 DLPG:PE ratio),followed by addition of 4 mU of PLA1; (C) fluorescence of the +2C/DLPGaggregates over the course of PLA2 activity with 1.4 μM PE and a DLPGconcentration of 7.27 μM, with enzyme added ranging from 0.5 to 5 mU ofPLA2; (D) 1.4 μM of +2C with DLPG at a series of concentrations from2.37 to 17.8 μM (1.7-12.7 DLPG:PE ratio), followed by addition of 40 mUof PLA1. t−−1 s is the time of enzyme addition.

As can be seen in FIGS. 8A-D, decomposition of the DLPG lipids by PLA1and PLA2 results in swift quenching of the fluorescence and increasedtransmittance at 440 nm. While monitoring either absorbance orfluorescence can allow one to determine enzyme activity, enhancedfluorescence quenching and dequenching allows for a more sensitivesensor to be achieved through fluorescence monitoring. In FIGS. 8B andD, the effects of varying lipid concentration on the rate of enzymaticdegradation was tested. In samples which had a lipid concentrationhigher than the saturation point of ˜1:16 PE:lipid ratio, a lag periodwas observed after the addition of the enzyme. This lag period is tiedto the amount of excess, “free” lipid in solution, as it increases withincreasing lipid and constant PE concentration. It is likely that thereis a population of lipids which are circulating in solution withoutbeing involved in an aggregate with a PE, and these lipids can act as asort of “reserve,” which can become involved in an aggregate, if needed.The enzymes will also be acting on these free lipids, halting thedegradation of the PE-Lipid sensor. Once this population of excesslipids is enzymatically cleaved by PLA1 or PLA2, the lipids making upthe sensor are then disassembled and the fluorescence quenching occurs.FIGS. 8A and C demonstrate the high sensitivity of the phospholipasesensor, as enzymatic cleavage is observed with both PLA1 and PLA2 atenzyme concentrations below 5 mU/mL. Since the weight of PLA1 from T.lanuginosus is not known, and the enzyme is obtained with concentrationlisted in terms of units of activity, it is difficult to compare PLA1limits of detection on a molar basis. The concentration of PLA2 thatcorresponds with 0.5 mU/mL at 320 U/mg protein is 500 fM, marking atleast a 10-fold increase in sensitivity over the previously reported PLCsensor.

Interestingly, the activity of PLC does not result in a strong change tothe sensors aggregated state. The changes resulting from addition of PLCwere studied, and it was clear that not only is there no rapidfluorescence quenching or absorbance change observed with PLC as wasseen with PLA1 and PLA2, but there is even a slight fluorescenceenhancement. This behavior suggests that one or both of the products ofPLC degradation, diacyl glycerol and 1-lauroyl-sn-glycerol 3-phosphate,also result in aggregation of the PE which allows retention of theenhanced fluorescence.

Example 2.8 Monitoring Enzyme Kinetics—PLA Sensing

The difference between the aggregation of PLA and AChE sensors isfurther shown by examining the kinetics of complex formation atdifferent concentrations. The ability to quantify substrateconcentration is afforded by these sensors when substrate concentrationcan be effectively calculated from the fluorescence or absorbance of theaggregate. It is visible when comparing FIGS. 6A and B with FIGS. 6C andD that the increase of the absorbance (430 nm) or fluorescence peaks(440 nm) representing the aggregate changes with a differentconcentration dependence for the two sensors. This is better shown viacalibration curves to fit fluorescence or absorbance to substrate (DLPGor LaCh) concentration.

The linear correlation between fluorescence at 440 nm and substrateconcentration is given for both PLA and AChE sensors in FIGS. 9A-C,showing linear regression of (A) absorbance of 10 μM PE-2+; (B)fluorescence of 1.4 μM PE-2+ (Ex: 375 nm, Em: 440 nm), with increasingDLPG concentration; (C) fluorescence of PE-1- with increasing LaChconcentration (Ex:370 nm, Em: 440 nm). The curves shown in FIGS. 9A-Cillustrate a difference in the concentration dependence for formation ofthe PLA and AChE sensor. +2C shows a linear increase in fluorescencewith increasing DLPG concentration, but −1C shows a sharp change with atypical sinusoidal shape between 20 and 30 μM of LaCh. The linearresponse of the PLA sensor is ideal for quantification of kineticparameters, as the concentration of lipid can be calculated from thelinear regression. The sinusoidal response of the AChE sensor doesprovide a linear fluorescence signal to LaCh concentration. Thissuggests that despite the similarities between the aggregates, theformation of the aggregates follows different kinetics. The kinetics ofthe degradation of the +2C/DLPG sensor by PLA1 and PLA2 were followed byconversion of the fluorescence or absorbance to concentration, asdiscussed above. An example of the result is given in FIGS. 10A-B, wherethe loss of fluorescence over time is converted into velocity vssubstrate concentration for calculating enzyme kinetics. FIGS. 10A-Billustrate (A) fluorescence of PLA sensor (Ex. 375 nm; Em. 440 nm)composed of 1.4 μM+2C and 16 μM DLPG following addition of 0.04 U ofPLA1; (B) velocity versus substrate plot after conversion of data in (A)to velocity and substrate following the equations given in the methodssection.

The activity of PLA1 and PLA2 were determined by nonlinear fitting offluorescence or absorbance of the aggregated PEs in the sensor by theHill fit. PLA1 from T. lanuginosus was found to have a V_(max) of141.7+/−6.8 μM/min, and a k_(m) of 5.41+/−0.28. PLA2 from C. adamanteusvenom had a V_(max) of 37.4+/−1.84 μM/min and a k_(m) of 6.39+/−0.29.The specific activity of PLA2 was calculated using 0.05 U/mL of 320 U/mgPLA2 to be 1295 μMol·min⁻¹ mg⁻¹, nearly 1000-fold greater than the 14μMol·min⁻¹ mg⁻¹ obtained from a previous study of PLA2 from C. atroxvenom. For these sensors the km is tied to the PE concentration, and incases with PLA1 where the PE concentration is 10 μM instead of 1.4 μM,the kM is 97 μM rather than 5.4. A strong correlation is observedbetween increased substrate concentration and enzyme activity due to acooperative effect. This is expressed as n in equation 2, which was fitto the results to determine kinetic parameters. In a case of nocooperativity, n is equal to one, but for both PLA1 and PLA2 it is fitto be 3. The cooperative effect is visible in FIG. 10B, where there is adecreased slope of v/[S] in regions of low substrate. This isreasonable, as PLA1 and PLA2 have been previously shown to bemembrane-associated proteins which have activity that is highlydependent on the local lipid environment.

Example 2.9 Monitoring Enzyme Activity—Acetylcholinesterase

Acetylcholinesterase (“AChE”) is an important enzyme which isresponsible for terminating synaptic transmission by hydrolyzing theneurotransmitter acetylcholine. In addition to the absorbance andfluorescence spectra in FIGS. 6A-D, the quantum yields were calculatedto be near unity upon formation of aggregates between −1C and lauroylcholine. This highly-sensitive fluorescence response in particular makesthis an ideal sensor for detection of AChE. The detection of AChE byfluorescence and absorbance using the −1C/LaCh sensor is shown in FIGS.11A-B, showing (A) absorbance at 430 nm and (B) fluorescence (Ex:370 nm,Em:440 nm) of −1C and LaCh at 0.2, 0.4, and 0.6 U of AChE.

As shown in FIGS. 11A-B, there is clear detection of AChE activitythrough the loss of the characteristic absorbance at 430 nm andfluorescence at 440 nm over time. There was a clear difference in therate of enzymatic degradation of the complex which correlated withamount of enzyme added. There is a slightly different profile to thechange in absorbance versus fluorescence over time. In the absorbancespectrum, there is a quick drop from 0.16 OD, and for the 0.4 and 0.2Unit additions of AChE this rate progressively decreases, leading to acurve with a more gradual slope. The slope of the 430 nm absorbance lossafter adding 0.6 Units of enzyme is fairly constant until 0.1 ODapproaches, indicating that the aggregate has been dissociated.Monitoring the enzyme activity through fluorescence gives similarresults as absorbance, except that the magnitude of change is greater.In FIG. 11B, the fluorescence drops an order of magnitude from 1E6 to1E5 photons/second, compared with a change from 0.16 to 0.1 OD for thechange in absorbance at the same concentration. The fluorescence spectrain FIG. 11B are similar to the absorbance spectra in FIG. 11A, and thechanges occur on the same timeframe. These results show that the−1C/LaCh sensor is effective at detection of AChE activity both throughcolorimetric means (absorbance) and through fluorescence assays.

As stated above, AChE is responsible for termination of nerve signals.This causes many inhibitors of AChE to be highly neurotoxic, and manypesticides and nerve agents are strong AChE inhibitors. In order todetermine whether the AChE sensor based on the −1C/LaCh complex could beused for detection of AChE inhibitors such as nerve agents andpesticides, the sensor was added to a solution of one of three differentAChE inhibitors prior to addition of AChE. While the compounds; TAE-1,Itopride, and Meptazinol, all have been shown to be AChE inhibitors,these compounds are less volatile and toxic than the nerve agents andpesticides that are of primary interest for AChE inhibition detection.The inhibition of AChE by these three inhibitors using the −1C/LaChsensor was carried out as described above, and the fluorescence of thesensor over time with and without inhibitor is given in FIGS. 12A-C,illustrating a −1C/LaCh complex (5 μM PE, 32 μM LaCh) showingfluorescence change after addition of 0.6 U of AChE in the presence ofAChE inhibitor (A) itopride HCl; (B) meptazinol HCl; and (C) TAE-1;traces with inhibitor are denoted with +I and the inhibitorconcentration, and −I indicates no inhibitor.

As shown in FIGS. 12A-C, it is clear that inhibition of AChE by severaldifferent inhibitors is apparent in the attenuated loss of fluorescencecompared with the reference solution, with no inhibitor. In order toconfirm that this result is not due to aggregation between the PE andthe inhibitors, the absorbance and fluorescence spectra of the PE andinhibitor without LaCh were obtained. FIGS. 13A-B illustrate (A)absorbance and (B) fluorescence (Ex: 370 nm) of 5 uM−1C with 5 ug/mL ofeither LaCh or one of the three AChE Inhibitors used in this study. Asshown in FIGS. 13A-B, there is no significant aggregation induced byItopride or Meptazinol. TAE-1 however, does result in a red-shiftedabsorbance and a strongly red-shifted and broadened green fluorescence.Further, there is little overlap between the fluorescence of theTAE-1/PE complex and that of the PE/LaCh complex.

Example 2.10 Prediction of Aggregate Structure by Molecular Simulations

To study the structure of the aggregate formed between cationic PEs andanionic phospholipids, simulations with +1C and DOPG near theexperimentally-observed ratio of lipid:PE were performed using all-atommolecular dynamics. While +2C was used primarily in this study, +1C wasshown to form an aggregate resulting in similar photophysical changes(FIGS. 14A-B, illustrating (A) absorbance and (B) fluorescence of 4.3uM+1C with various concentrations of DLPG). In order to reducecomputational time, +1C was used in the simulations rather than +2C. Itshould be noted that the simulations where only 2 PEs were used did notresult in the formation of an aggregate within the 150 ns simulationtime, as the two PEs in the simulation never came close enough tointeract with one another in this time. This was observed at bothsimulation box sizes used (8 nm or 10 nm side length), and with a PElipid ratio of 1:3 and 1:10. Timelines of the three simulations in whicha

Example 3 Example 3.1 Overview

Four PEs (Scheme 3) were synthesized for evaluation against native henegg white lysozyme (HEWL) amyloids. The PEs used, designated for brevityPEn+ and PE1−, all have ethyl ester terminal moieties on the PE backboneand side-pendant charged groups; the cationic compounds have n=1, 2 and3 repeat units and the anionic compound has one repeat unit. Thecompounds are amphiphilic and water soluble due to the hydrophobicbackbone and charged side groups. These ester-terminated compounds wereselected for the effective sensing modality of fluorescence yieldincrease from reduced quenching by water when bound to a hydrophobicsurface.

Example 3.2 Formation and Characterization of HEWL Amyloids

Hen egg white lysozyme (HEWL) was used to form fibrillary amyloidaggregates for use in this study. Lysozyme has been suggested as auseful model protein for amyloid studies, due to its low cost and therelative ease with which it can be induced to form amyloid aggregates.Lysozyme amyloid oligomers and fibrils have also been shown to exhibitcytotoxicity towards human neuroblastoma cells, indicating that theamyloid-aggregate conformer of lysozyme recapitulates most of therelevant properties of known disease-associated proteins. For theseexperiments, HEWL (Sigma-Aldrich) was incubated at 70° C. and aconcentration of 350 μM in pH 3 sodium citrate buffer (10 mM) with 100mM NaCl. Visible precipitates of aggregated lysozyme were observed toaccumulate over the time of incubation, and the formation of amyloidfibrils was determined by Thioflavin T (ThT) fluorescence assay (FIG.15, illustrating a plot of PE and Thioflavin T (10 μM) fluorescenceenhancement with variously incubated HEWL (10 μM monomer basis, 0.5mg/mL), with unbound dye fluorescence normalized to 0 and maximum dyefluorescence normalized to 1), far-UV circular dichroism (CD)spectroscopy (FIG. 16, illustrating far-UV circular dichroism spectra of0 h, 2 h and 4 h incubated HEWL (0.14 mg/mL) in pH 3 citrate buffer (10mM)), and atomic force microscopy (AFM) and transmission electronmicroscopy (TEM) (FIG. 17, illustrating TEM (top) and AFM (bottom)images of 0 h, 1 h, 1.5 h and 4 h incubated HEWL; scale bars=200 nm; 4h, inset: view of a single isolated fibril, showing twisted morphology;AFM image Z-height: 0 h, 25 nm; 1 h, 25 nm; 1.5 h, 15 nm; 4 h, 100 nm).

ThT-positive aggregates were detected by the second hour of incubation(FIG. 15), and the profile of ThT fluorescence enhancement overincubation time had the sigmoidal shape consistent with thenucleation-dependent mechanism that is well accepted for amyloidformation. Far-UV circular dichroism measurements (FIG. 16) showedconversion of primarily α-helix structure of monomeric lysozyme (0 h),as indicated by the negative bands at 222 and 208 nm, into primarilyβ-sheet structure in the mature aggregates (2 h and 4 h), as indicatedby the single negative band at 218 nm and the positive band just visibleat the 200 nm edge of the spectrum.

Fibrillar morphology of HEWL aggregates was confirmed by directvisualization by AFM and TEM. AFM on dry mica and TEM (FIG. 17) onnon-glow discharged carbon grids showed that unincubated HEWL formed ahomogeneous film without large features. One hour of incubation causedthe HEWL to form distinguishable bumps, hypothesized to bepre-thioflavinophilic oligomers. By 1.5 hours of incubation when amyloidformation was just reaching plateau phase as indicated by ThTfluorescence, small linear aggregates were observed, which lengthened bythe fourth hour into short, bundled fibrils 20-30 nm wide and 60-200 nmlong. No fibrils significantly longer than these were observed, even forlonger incubated samples. These fibrillary, β-sheet enriched,ThT-positive HEWL amyloid aggregates were then used to evaluate thebinding activity and photophysical changes of PEs against amyloid.

Example 3.3 Spectrophotometry of PE-HEWL Interactions

Excitation and emission spectra of PEs in phosphate buffer alone, withmonomeric HEWL and with HEWL amyloids (8 h incubated) are shown in FIGS.18A-H, and relevant photophysical properties are summarized in Table 1.FIGS. 18A-H illustrate excitation (A, B, C, D) and emission (right; E,F, G, H) spectra of PEs (A, E: PE1+; B, F: PE1−; C, G: PE2+; D, H: PE3+)in phosphate buffer (PB, pH 7.4, 10 mM) alone (long dashed line) withHEWL monomers (short dashed line) and with HEWL amyloids (solid line);PE concentration: 500 nM, protein concentration: 5 μM monomer basis/0.25mg/mL; emission and excitation wavelengths, respectively, were chosen asshown in Table 1 for each sample. A 10:1 molar ratio of protein to PEwas used for these experiments. Absorbance spectra were taken, butbackground light scattering from insoluble amyloid aggregates made themdifficult to interpret, so “fluorescence detected absorbance” in theform of excitation spectra was used instead. Normalized excitation andemission spectra, in which peak shifts and lineshape changes of spectrawere somewhat easier to visualize, were used. All four PEs exhibitedsignificant fluorescence enhancement in solution with HEWL amyloids(FIGS. 18E, F, G, H), and no fluorescence change with HEWL monomersexcept for PE1−. The fluorescence enhancement over baseline was mostsignificant for the longer PE2+ and PE3+ (FIGS. 18G and H), which alsohad notably sharpened fluorescence spectra with small (˜10 nm)blueshifting of the maximum. PE1− had a similarly sharpened andblueshifted emission spectrum (FIG. 18F) with both HEWL monomers andamyloid, with the addition of a shoulder at 465 nm with amyloid. PE1+(FIG. 18E) had no change in wavelength or lineshape of emissionspectrum, just a large increase in intensity when mixed with amyloids.The excitation spectra (FIGS. 18A, B, C, D) show a notable bathochromicshift for each PE in solution mixed with amyloid, of 23, 27, 35 and 29nm for PE1+, PE2+, PE3+ and PE1−, respectively, observing only thelow-energy band. The high-energy band, less relevant for imagingpurposes, was also bathochromically shifted. The cationic PEs, asbefore, did not appear to interact with HEWL monomer in such a way as toproduce a fluorescence change. PE1− had similar excitation spectrum(FIG. 18B) with monomer and amyloid, except for a large intensitydifference.

A plot of normalized fluorescence enhancement for all four PEs andThioflavin T with HEWL fibrils incubated for different lengths of timeis shown in FIG. 15. PE1+ and PE2+ track amyloid formation in roughlythe same way as ThT, showing a sigmoidal curve with onset of alogarithmic growth phase occurring at the same time, around 1 hour ofincubation. The plateau phase, as monitored by fluorescence of any ofthose three PEs or ThT, appeared at 2 hours incubation. PE3+fluorescence enhancement shows a similar length lag phase but a slowergrowth phase, taking up to 3.5 hours to reach its plateau phase. PE1−has greater fluorescence enhancement when mixed with monomeric speciesthan with amyloid at the equimolar concentrations used for this assay.

Example 3.4 Determination of PE/Amyloid Binding Constants

Next, binding saturation assays were conducted to quantify the affinityof PE-amyloid binding; data and fitted curves were produced and thefitted parameters are summarized in the last two columns of Table 1.Since the linear fibril binding sites could fit many PEs, fits tobinding curves were performed to the Hill equation to capture possiblebinding cooperativity:

$y = \frac{F_{\max}x^{n}}{K^{n} + x^{n}}$

where x is PE concentration (with protein concentration fixed), y is PEfluorescence intensity, F_(max) is PE fluorescence intensity atsaturation, k is the equilibrium dissociation constant and theexponential term n is the Hill parameter which describes cooperativityof binding. n=1 indicates non-cooperative, independent binding, n>1indicates that binding of one ligand increases affinity of the bindingof a second, and n<1 indicated that binding of one ligand decreasesaffinity of the binding of a second. Fits to the data for the threecationic PEs produced F_(max) values close to the observed saturationvalue, and the other two parameters are reported as calculated. The fitsindicated that PE1+, PE2+ and OPE3+ bound to HEWL amyloid with lowmicromolar affinity dependent on PE length, with the calculateddissociation constant decreasing from 2.6 μM for PE1+ to 1.15 μM forPE2+, and still further to 858 nM for PE3+. Furthermore, thecooperativity of PE binding increased from almost no cooperativity forPE1+, to some positive cooperativity for PE2+, to still more positivecooperativity for PE3+. The quantitative physical meaning of the Hillparameter is not quite clear except in special cases, but in a generalway it is possible to conclude that for the cationic PEs, the shortestPE has non-cooperative binding to HEWL amyloid (Michaelis-Mentenbinding), and the two longer PEs have increasingly positivelycooperative binding. The binding of PE1− to HEWL monomers appeared to belinear and non-saturable at reasonable concentrations (see, FIG. 19,illustrating linear (non-saturable) binding of PE1− to HEWL monomers (5μM/0.25 mg/mL); linear fit shown for clarity; this experiment wasperformed once), indicating a low-affinity binding to a very largenumber of sites. The effect of PE1− non-specific binding to HEWLmonomers precluded accurate determination of a binding constant forPE1−/amyloid interactions such that a quantitative comparison of bindingbetween the cationic and anionic compounds could not be made.

TABLE 1 Relevant photophysical properties of PEs alone and bound to HEWLamyloid, and apparent binding constants and Hill coefficients of PEbinding to HEWL amyloid. λ_(ex) λ_(em) λ_(ex) (PB λ_(em) λ_(em) (PB w/φ_(fl) φ_(fl) (PB) w/HEWL) (H₂O) (PB) HEWL) K_(d) Hill Comp. (H₂O)(MeOH) (nm) (nm) (nm) (nm) (nm) (μM) coeffi. PE1+ 0.023 0.75 314, 362332, 385 454 454 454 2.63 ± 0.58 1.04 PE2+ 0.039 0.71 330, 399 337, 426448 460 445 1.15 ± 0.26 1.15 PE3+ 0.069 0.70 340, 399 335, 434 440 464453 0.858 ± 0.058 1.89 PE1− — — 314, 370 327, 399 454 454 439 — —

Example 3.5 Induced Circular Dichroism of PE-Amyloid Complexes

Circular dichroism measurements were performed to determine if theintrinsic chirality of the HEWL fibrils was transferred to the PEchromophore by a chiral backbone twist or an “excitonic” chiralsupramolecular aggregate. CD spectroscopy (CPEs in PB with HEWL monomerand with HEWL amyloid; performed for (a): PE1+; (b): PE1−; (c): PE2+;(d): PE3+; PEs 10 μM, HEWL 10 μM monomer basis/0.5 mg/mL) indicatedinduced circular dichroism of PEs when bound to HEWL amyloids. Asexpected, no PE had optical activity by itself in phosphate buffersolution and none of the PEs had any CD with HEWL monomer, includingPE1−. PE1+ did not have optical activity with HEWL amyloid, but theother three PEs did. PE1−, PE2+ and PE3+ all had strong induced CD witha negative Cotton effect when bound to HEWL amyloid fibrils. PE2+ andPE3+ gave rise to similar CD spectra, with more intense bands in thespectrum for PE3+. The induced CD spectrum for PE1− had a pronouncedtwo-band structure, reflecting the more intense high-energy band for theanionic PE when bound to HEWL amyloid.

Example 3.6 Protein→PE Energy Transfer in PE-Amyloid Complexes

Since lysozyme is an intrinsically fluorescent protein whose emissionspectrum overlaps significantly with the excitation spectra of PEs, wechose to investigate the possibility of Förster resonance energytransfer (FRET) from the protein chromophore to PEs by a simplespectroscopic method. Emission spectra of solutions containing PEs andHEWL amyloids or monomers were obtained using the excitation wavelengthof HEWL (280 nm) and PE emission was observed only from PE/amyloidsamples, indicating that HEWL→PE energy transfer was occurring only withPEs bound to amyloid fibrils. The results are summarized as FRETefficiencies in FIG. 20, illustrating HEWL→PE FRET efficienciescalculated from spectral data by the equation E=F_(A)/(F_(D)+F_(A))where F_(D) is the integrated area under the donor emission peak, andF_(A) is the integrated area under the acceptor emission peak. Thissimple expression is valid for this case since the PEs arenonfluorescent when excited at the donor excitation wavelength,eliminating crosstalk. Thus, F_(A) is the total number of energytransfer events, and (F_(A)+F_(D)) is the total number of excitationevents. Theoretically, the efficiencies should be convertible intodistances by

${E = \frac{1}{1 + \left( \frac{r}{R_{0}} \right)^{6}}},$

but PEs and HEWL amyloids are not a well-characterized FRET pair with adefined Förster radius R₀. Qualitiatively, some determinations based onthe relative measured efficiencies for the different PEs can be made.The measured FRET efficiency will be affected by multiple independentfactors averaged over all the PE-HEWL pairs in solution, such as thenumber of bound PE molecules, the bound PE-HEWL chromophore distance,and the spectral overlap integral J(λ), all of which will vary by PE.The highest efficiency observed for PE2+ is probably the result of itshigher binding constant than PE1− or PE1+ combined with its greateroverlap integral than PE2+.

Example 3.7 Explicating the Mode of PE-Amyloid Binding

The results of these experiments indicate modes of interaction betweenPEs and HEWL monomers and amyloids, as summarized in cartoon form inFIG. 21. All four PEs tested were observed to bind to HEWL amyloid, withgood affinity, but with different properties depending on chain lengthand charge. Generally, the PEs either do not interact with protein, bindas single molecules, or bind as J dimers which are either racemic orchirally biased. Overall, it has become clear that J-type aggregation isa naturally favorable mode of PE-PE interaction for PEs with side chaincharged groups when the Coulombic repulsion between the charged groupsis reduced.

The spectral changes of PE2+, PE3+, and PE1− in complex with HEWLamyloids, and of PE1− in complex with HEWL monomers, are highlyindicative of J aggregation: redshifted absorbance, sharpening offluorescence band, and narrowed Stokes shift. The enhancement offluorescence intensity is attributable all or in part to the reducedquenching of the PE by water when bound to the hydrophobic surface ofthe protein; this solvent-access effect is in play for all four PEs. Thecurrent study indicates that the longer cationic PEs. PE2+ and PE3+,form J dimers (or possibly larger aggregates) on the HEWL amyloid fibrilsurface, and PE1− forms J dimers on both HEWL monomers and HEWL amyloidfibrils. The aggregates formed on amyloid have a chiral bias to thePE-PE offset angle, producing a chiral supramolecular chromophore, or anexcitonic optical activity, responsible for the circular dichroism seenexperimentally. The exact source of this bias is hard to pin down; itcould be a result of the helically twisted fibril axis or more specificto a binding site. Notably, the aggregates formed by PE1− must beracemic, indicating that the PEs are not interacting with a specificsite but simply sticking to oppositely charged areas of the lysozymesurface. PE1+ has some small redshifting of excitation spectrum, but itsemission spectrum does not shift at all and it acquires no opticalactivity, indicating that this compound binds to HEWL fibrils as singlemolecules rather than as a structured aggregate. The small excitationredshift could be due to minor backbone planarization, and the increaseof fluorescence intensity to reduced solvent access.

The results of binding saturation assays support these conclusions forthe cationic PEs. PE2+ and PE3+ show positive cooperativity, meaningthat the binding of one PE increases the affinity of the next bindingevent. The formation of J aggregates on the fibril surface satisfiesthis condition: a single PE might bind to a favorable site, and a secondfinds it and forms an even more favorable J aggregate due to π-π andhydrophobic interactions. It is also possible that PEs could form Jdimers in solution that subsequently find the fibril surface, but thisseems unlikely due to charge repulsion. Furthermore, the curve for PE3+shows a larger cooperative effect than that for PE2+, and the induced CDbands for PE3+ are also more intense; the increased length of PE3+increases the available area for aggregate formation, forming more orlarger chiral aggregates. The PE1+/HEWL amyloid binding assay indicatedno cooperative binding effect, which is consistent with independent,single-molecule binding.

One notable result of this study is the large differences between PE1+and PE1− in their interactions with HEWL monomers and amyloid. The twosingle-repeat PEs tested differ by the charge on the side-pendantsolubilizing groups, and their interactions with HEWL monomer andamyloids were highly different. PE1+ exhibited non-cooperative andsaturable binding to amyloid without induced optical activity or largeshifts in absorption or emission bands, and when bound, its emission wasthe least enhanced over free PE. Its anionic counterpart, PE1−, provedquite different both in its nonspecific binding to HEWL monomer and inits interaction with HEWL amyloid. This interaction was seen to befairly weak, as indicated by the non-saturable binding, but itoverwhelms the PE1−/amyloid interaction at high PE concentrations, afterall the amyloid binding sites are occupied. In vitro, withoutinterfering effects from other cellular/tissue components, such aneffect could prove useful for monitoring the disappearance of similarlycharged monomers. The differences in the PE1+ and PE1− binding toamyloid-PE1+ binds singly and PE1− as chiral J aggregates—could be dueto charge or H-bonding interactions specific to sites on the lysozymefibril surface. The specificity of these possible charge effects isnotable, since the arrangement of charged residues on the fibril surfaceis controlled by the protein's primary, secondary and tertiarystructure. This effect may provide useful means of differentiatingamyloids formed from different monomers.

The absence of FRET in any monomer/PE solution reconfirms the weak andnon-specific nature of PE1−/monomer binding, since the PE is not heldwithin range of the fluorescing residues. Aromatic residues may alsoonly be surface exposed, and within range of transfer to PEs, in theamyloid state; HEWL intrinsic fluorescence is found to decrease over thecourse of incubation, which implies that fluorescing residues areincreasingly exposed to solvent as more amyloid forms. The largedifferences between PE1+ and PE1−'s interactions with monomeric andamyloid HEWL, likely influenced by specific charged residues in the HEWLprimary sequence, may be employed for differentiation of amyloids withdifferent monomers, a useful effect, for example, in the study of theintermediate disorder called dementia with Lewy bodies, in which Lewybodies (formed of alpha-synuclein and normally characteristic ofParkinson's disease) and amyloid plaques are comorbid.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theembodiments of the present invention. Thus, it should be understood thatalthough the present invention has been specifically disclosed byspecific embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those of ordinaryskill in the art, and that such modifications and variations areconsidered to be within the scope of embodiments of the presentinvention.

Example 3.8 PE Synthesis and Chemical Reagents

Except PEs, all reagents were obtained commercially and used withoutfurther purification. Synthesis of PEs has been reported previously,except for PE1−, which was synthesized analogously to PE1+. Hen eggwhite lysozyme and Thioflavin T were obtained from Sigma-AldrichChemical Company (St. Louis, Mo.). Suspensions of protein aggregateswere gently vortexed to distribute aggregates before use in experiments.

Preparation of Lysozyme Amyloid Fibrils.

Lyophilized HEWL was dissolved at 10 mg/mL in 10 mM pH 3 sodium citratebuffer with 0.1 M NaCl. The solution was incubated in a 70 C oil bathand magnetically stirred at 250 rpm for 12 h, and aliquots werewithdrawn at half-hour intervals. The initially clear solution wasobserved to form cloudy aggregates by 1 h incubation. Half of eachaliquot was immediately diluted into pH 7.4 phosphate buffer to preventfurther influence of acidic conditions and stored at 4 C. The sampleswere observed to undergo no noticeable degradation over the course ofone month, and these neutralized aliquots were used for all followingexperiments except for measurements of protein circular dichroism.

Spectrophotometry of PE/ThT-protein Complexes.

For studies of fluorescence enhancement vs. protein incubation time,dyes were mixed with protein sample in phosphate buffer (10 mM, pH 7.4)at equal monomer concentration of 10 μM in the wells of a standard96-well plate. Emission spectra were obtained using top read with aSpectraMax M2e plate-reading spectrophotometer (Molecular Devices,Sunnyvale, Calif.). Experiments were performed in duplicate and errorsare reported as standard deviation. For analysis of bound PE excitationand emission spectra and protein-PE energy transfer, PEs (500 nM) weremixed with protein sample (5 μM, monomer basis) in phosphate buffer andthe solution transferred to a quartz fluorometry cuvette. Spectra wereobtained on a PTI QuantaMaster 40 steady state spectrofluorometer(HORIBA Scientific, Edison, N.J.).

Circular Dichroism Spectroscopy.

PEs and protein samples were diluted in phosphate buffer, gentlyvortexed, and read in a 1 mm pathlength quartz CD cuvette using an Aviv410 CD spectrometer (Aviv Biomedical, Lakewood, N.J.), 15 s averagingtime. A blank spectrum (PB only) was subtracted from each sample toremove background activity. Error bars are standard deviation overmultiple reads of a single sample as reported by the instrument.

Determination of Binding Constant.

For determination of binding constant of PEs to amyloid aggregates, PEswere mixed with HEWL amyloid in phosphate buffer at a finalconcentration of 100 nM-5 μM for PEs and 5 μM (monomer basis) forprotein. The solutions were then transferred to a quartz fluorometrycuvette and emission measured at the pertinent wavelength. Experimentswere performed in duplicate and errors reported as standard deviation.Hill function fits to PE binding curves were calculated in OriginPro 9.

AFM Imaging.

For AFM, a droplet of each protein sample at 5 mg/mL was pipetted ontofreshly cleaved mica substrate and allowed to physisorb for 20 min,followed by a single rinse with HPLC-grade water and gentle drying undera stream of N₂. Imaging was performed with a Nanoscope IIIa AFM (Veeco,Plainview, N.Y.) in tapping mode under a constant stream of dry N₂ gasusing a rectangular silicon cantilever with a spring constant of 40 N/m(Veeco model RTESPA-W). Veeco Nanoscope software was used to capture andanalyze the images. 0 h and 1 h images are cropped from 1 μm widthimages subjected to a first-order x,y plane fit and flattened. 1.5 himage is cropped from a 5 μm width image subjected to a third-order x,yplane fit and flattened. 4 h image is cropped from a 5 μm width imagesubjected to a first-order x,y plane fit.

TEM Imaging.

For TEM imaging, incubated HEWL solutions at a concentration of 350 μMwere diluted 1:5 in water and aliquoted onto carbon-coated grids,allowed to adsorb, washed with deionized water and stained with 2%uranyl acetate solution. Excess liquid was removed and the samplesallowed to dry in air. Samples were imaged on a Hitatchi H7500transmission electron microscope (Hitachi High Technologies Corp.,Tokyo, Japan) with tungsten filament illumination, operating with an AMTX60 bottom mount CCD camera detector.

Additional Embodiments

The following exemplary embodiments are provided, the numbering of whichis not to be construed as designating levels of importance:

Embodiment 1 provides a method of inducing germination of microbialspores comprising contacting the microbial spores with a p-phenyleneethynylene compound.

Embodiment 2 provides the method of Embodiment 1, wherein the microbialspore is at least one of a Bacillus anthracis, a Bacillus atrophaeus, aBacillus cereus, and a Bacillus subtilis.

Embodiment 3 provides the method of any one of Embodiments 1-2, whereinthe p-phenylene ethynylene compound comprises a repeating unit havingthe structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;

at each occurrence R² is independently chosen from —H, (C₁-C₅)alkyl,—SO₂ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, phenolate,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, and—N⁺(R^(A))₃ wherein at each occurrence R^(A) is independently(C₁-C₅)alkyl;

at each occurrence L¹ is independently chosen from a bond and

and

j is about 0 to about 4.

Embodiment 4 provides the method of any one of Embodiments 1-3, whereinthe p-phenylene ethynylene compound comprising a repeating unit havingthe structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

and

n is about 2 to about 4.

Embodiment 5 provides the method of any one of Embodiments 1-4, whereinthe p-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4;

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, —C(O)NH—(C₁-C₁₀)hydrocarbyl,and C(O)OH; and

m is about 1 to about 1,000.

Embodiment 6 provides the method of Embodiment 5, wherein thep-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 50.

Embodiment 7 provides the method of Embodiment 6, wherein A is—C(O)OCH₂CH₃.

Embodiment 8 provides the method of any one of Embodiments 1-7, whereinthe p-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl; and

p is about 1 to about 10.

Embodiment 9 provides the method of Embodiment 8, wherein

at each occurrence Z is —O—;

at each occurrence L² is independently (C₁-C₅)alkyl;

at each occurrence R² is independently chosen from —N⁺(CH³)₃,

and

p is about 1 to about 5.

Embodiment 10 provides the method of any one of Embodiments 8-9, whereinthe p-phenylene ethynylene compound has the structure:

wherein p is about 2.

Embodiment 11 provides a method for detecting an enzyme, the methodcomprising (i) introducing an enzyme to a composition comprising ap-phenylene ethynylene compound and an enzyme substrate; and (ii)analyzing the change in fluorescence of the p-phenylene ethynylenecompound following introduction of the enzyme.

Embodiment 12 provides the method of Embodiment 11, wherein thep-phenylene ethynylene compound and the enzyme substrate form a complex.

Embodiment 13 provides the method of any one of Embodiments 11-12,wherein the introduction step (i) and the analyzing step (ii) occur inan aqueous environment.

Embodiment 14 provides the method of Embodiment 12, wherein thefluorescence of the p-phenylene ethynylene compound decreases followingthe introduction of the enzyme.

Embodiment 15 provides the method of Embodiment 14, wherein thefluorescence decreases due to a molecular transformation of the enzymesubstrate to an entity or entities that do not complex with thep-phenylene ethynylene compound.

Embodiment 16 provides the method of any one of Embodiments 11-15,wherein the p-phenylene ethynylene compound is a cationic p-phenyleneethynylene compound.

Embodiment 17 provides the method of Embodiment 16, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4;

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and

m is about 1 to about 10.

Embodiment 18 provides the method of any one of Embodiments 16-17,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 19 provides the method of any one of Embodiments 16-18,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein t is about 1 to about 3.

Embodiment 20 provides the method of any one of Embodiments 16-19,wherein the enzyme substrate is an anionic enzyme substrate.

Embodiment 21 provides the method of Embodiment 20, wherein the anionicenzyme substrate is 1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

Embodiment 22 provides the method of any one of Embodiments 11-21,wherein the p-phenylene ethynylene compound is an anionic p-phenyleneethynylene compound.

Embodiment 23 provides the method of Embodiment 22, wherein the anionicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and            phenolate;

j is about 0 to about 4:

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl,

m is about 1 to about 10.

Embodiment 24 provides the method of any one of Embodiments 22-23,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —SO₃ ⁻, —CO₂ ⁻,—H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate;

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 25 provides the method of any one of Embodiments 22-24,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein t is about 1 to about 3.

Embodiment 26 provides the method of any one of Embodiments 22-25,wherein the enzyme substrate is a cationic enzyme substrate.

Embodiment 27 provides the method of Embodiment 26, wherein the cationicenzyme substrate is a substituted or unsubstituted(C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.

Embodiment 28 provides the method of any one of Embodiments 26-27,wherein the cationic enzyme substrate is a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃.

Embodiment 29 provides the method of any one of Embodiments 26-28,wherein the cationic enzyme substrate is chosen from lauroyl choline andacetylcholine.

Embodiment 30 provides the method of any one of Embodiments 26-29,wherein the cationic enzyme substrate is lauroyl choline.

Embodiment 31 provides the method of any one of Embodiments 11-30,wherein the enzyme is chosen from phospholipase A1, phospholipase A2,phospholipase C, and acetyl cholinesterase.

Embodiment 32 provides a method for detecting an enzyme, the methodcomprising (i) introducing an enzyme to a composition comprising ap-phenylene ethynylene compound and an enzyme substrate; and (ii)analyzing the change in fluorescence of the p-phenylene ethynylenecompound following introduction of the enzyme;

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein s is about 1 to about 3;

the enzyme substrate is1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol); and

the enzyme is at least one of phospholipase A1, phospholipase A2, andphospholipase C.

Embodiment 33 provides a method for detecting an enzyme, the methodcomprising (i) introducing an enzyme to a composition comprising ap-phenylene ethynylene compound and an enzyme substrate; and (ii)analyzing the change in fluorescence of the p-phenylene ethynylenecompound following introduction of the enzyme:

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein t is about 1 to about 3;

the enzyme substrate is lauroyl choline; and

the enzyme is acetylcholinesterase.

Embodiment 34 provides a sensor, the sensor comprising a p-phenyleneethynylene compound and an enzyme substrate.

Embodiment 35 provides the sensor of Embodiment 34, wherein thep-phenylene ethynylene compound is a charged p-phenylene ethynylenecompound and the enzyme substrate is an oppositely charged enzymesubstrate.

Embodiment 36 provides the sensor of any one of Embodiments 34-35,wherein the p-phenylene ethynylene compound is a cationic p-phenyleneethynylene compound.

Embodiment 37 provides the sensor of Embodiment 36, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4:

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and

m is about 1 to about 10.

Embodiment 38 provides the sensor of any one of Embodiments 36-37,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 39 provides the sensor of any one of Embodiments 36-38,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein s is about 1 to about 3.

Embodiment 40 provides the sensor of any one of Embodiments 36-39,wherein the enzyme substrate is an anionic enzyme substrate.

Embodiment 41 provides the sensor of Embodiment 40, wherein the anionicenzyme substrate is 1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

Embodiment 42 provides the sensor of any one of Embodiments 34-41,wherein the p-phenylene ethynylene compound is an anionic p-phenyleneethynylene compound.

Embodiment 43 provides the sensor of Embodiment 42, wherein the anionicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and            phenolate;

j is about 0 to about 4:

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl,

m is about 1 to about 10.

Embodiment 44 provides the sensor of any one of Embodiments 42-43,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —SO₃ ⁻, —CO₂ ⁻,—H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate;

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 45 provides the sensor of any one of Embodiments 42-44,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein t is about 1 to about 3.

Embodiment 46 provides the sensor of any one of Embodiments 42-45,wherein the enzyme substrate is a cationic enzyme substrate.

Embodiment 47 provides the sensor of Embodiment 46, wherein the cationicenzyme substrate is a substituted or unsubstituted(C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.

Embodiment 48 provides the sensor of any one of Embodiments 46-47,wherein the cationic enzyme substrate is a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃.

Embodiment 49 provides the sensor of any one of Embodiments 46-48,wherein the cationic enzyme substrate is chosen from lauroyl choline andacetylcholine.

Embodiment 50 provides the sensor of any one of Embodiments 46-49,wherein the cationic enzyme substrate is lauroyl choline.

Embodiment 51 provides the sensor of any one of Embodiments 34-50,wherein the enzyme is chosen from phospholipase A1, phospholipase A2,phospholipase C, and acetyl cholinesterase.

Embodiment 52 provides a sensor, the sensor comprising a cationicp-phenylene ethynylene compound and an anionic enzyme substrate;

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein s is about 1 to about 3; and

the anionic enzyme substrate is1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

Embodiment 53 provides a sensor, the sensor comprising a p-phenyleneethynylene compound and an enzyme substrate;

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein t is about 1 to about 3; and

the enzyme substrate is lauroyl choline.

Embodiment 54 provides a method for protein analysis, the methodcomprising (i) introducing a p-phenylene ethynylene compound to abiological sample comprising at least one protein and (ii) analyzing thefluorescence of the p-phenylene ethynylene compound in the presence ofthe biological sample comprising the at least one protein.

Embodiment 55 provides the method of Embodiment 54, wherein thefluorescence of the p-phenylene ethynylene compound is analyzed prior tobeing introduced to the biological sample comprising the at least oneprotein.

Embodiment 56 provides the method of any one of Embodiments 54-55,wherein analyzing the fluorescence of the p-phenylene ethynylenecompound in the presence of the biological sample comprising the atleast one protein comprises analyzing the spectral changes between thefluorescence of the p-phenylene ethynylene compound of step (i) and thefluorescence of the p-phenylene ethynylene compound in the presence ofthe biological sample comprising the at least one protein of step (ii).

Embodiment 57 provides the method of any one of Embodiments 54-56,wherein the morphology of the protein in the biological sample isdetermined by analyzing spectral changes between the fluorescence of thep-phenylene ethynylene compound of step (i) and the fluorescence of thep-phenylene ethynylene compound in the presence of the biological samplecomprising the at least one protein of step (ii).

Embodiment 58 provides the method of Embodiment 57, wherein the spectralchanges in the fluorescence of the p-phenylene ethynylene compoundbetween step (i) and step (ii) are induced by changes in theconformational freedom of the p-phenylene ethynylene compound betweenstep (i) and step (ii).

Embodiment 59 provides the method of any one of Embodiments 54-58,wherein the p-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4;

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and

m is about 1 to about 10.

Embodiment 60 provides the method of any one of Embodiments 54-59,wherein the p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 61 provides the method of any one of Embodiments 54-60,wherein the p-phenylene ethynylene compound has the structure:

wherein s is about 1 to about 3.

Embodiment 62 provides the method of any one of Embodiments 54-61,wherein the protein is at least one of an amyloid beta protein, Aβ-40,Aβ-42, tau, and α-synuclein, islet amyloid precursor protein,Huntingtin, prion, lysozyme, TDP-43 (transactive response DNA-bindingprotein 43), FUS (fused in sarcoma) and insulin.

Embodiment 63 provides a method for protein analysis, the methodcomprising (i) analyzing the fluorescence of a p-phenylene ethynylenecompound; (ii) introducing the p-phenylene ethynylene compound to abiological sample comprising at least one protein; (iii) analyzing thefluorescence of the p-phenylene ethynylene compound in the presence ofthe biological sample comprising the at least one protein; and (iv)determining the morphology of the at least one protein in the biologicalsample by analyzing spectral changes between the fluorescence of thep-phenylene ethynylene compound of step (i) and the fluorescence of thep-phenylene ethynylene compound in the presence of the biological samplecomprising the at least one protein of step (iii);

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein s is 1; and

the protein is at least one of an amyloid beta protein, Aβ-40, Aβ-42,tau, and α-synuclein, islet amyloid precursor protein, Huntingtin,prion, lysozyme, TDP-43 (transactive response DNA-binding protein 43),FUS (fused in sarcoma) and insulin.

Embodiment 64 provides a method for detecting a chemical agent, themethod comprising (i) exposing a sensor composition comprising a complexcomprising a p-phenylene ethynylene compound and an enzyme substrate toa sample; (ii) introducing an enzyme to the sensor composition of step(i); and (iii) analyzing the change in fluorescence of the p-phenyleneethynylene compound between the exposing step (i) and the introducing anenzyme step (ii).

Embodiment 65 provides the method of Embodiment 64, wherein a change influorescence between the exposing step (i) and the introducing an enzymestep (ii) indicates the presence of a chemical agent that does interactwith the enzyme.

Embodiment 66 provides the method of any one of Embodiments 64-65,wherein a minimal change in fluorescence between the exposing step (i)and the introducing an enzyme step (ii) indicates the presence of achemical agent that does interact with the enzyme.

Embodiment 67 provides the method of any one of Embodiments 64-66,wherein the p-phenylene ethynylene compound is a cationic p-phenyleneethynylene compound.

Embodiment 68 provides the method of Embodiment 67, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4;

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and

m is about 1 to about 10.

Embodiment 69 provides the method of any one of Embodiments 67-68,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 70 provides the method of any one of Embodiments 67-69,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein s is about 1 to about 3.

Embodiment 71 provides the method of any one of Embodiments 67-70,wherein the enzyme is chosen from phospholipase A1, phospholipase A2,and phospholipase C.

Embodiment 72 provides the method of any one of Embodiments 67-71,wherein the enzyme substrate is an anionic enzyme substrate.

Embodiment 73 provides the method of Embodiment 72, wherein the anionicenzyme substrate is 1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

Embodiment 74 provides the method of any one of Embodiments 64-73,wherein the p-phenylene ethynylene compound is an anionic p-phenyleneethynylene compound.

Embodiment 75 provides the method of Embodiment 74, wherein the anionicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and            phenolate;

j is about 0 to about 4;

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl,

m is about 1 to about 10.

Embodiment 76 provides the method of any one of Embodiments 74-75,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently selected from —SO₃ ⁻, —CO₂ ⁻,—H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate;

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 77 provides the method of any one of Embodiments 74-76,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein t is about 1 to about 3.

Embodiment 78 provides the method of any one of Embodiments 74-77,wherein the enzyme substrate is a cationic enzyme substrate.

Embodiment 79 provides the method of Embodiment 78, wherein the cationicenzyme substrate is a substituted or unsubstituted(C₁₋C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.

Embodiment 80 provides the method of any one of Embodiments 78-79,wherein the cationic enzyme substrate is a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃.

Embodiment 81 provides the method of any one of Embodiments 78-80,wherein the cationic enzyme substrate is chosen from lauroyl choline andacetylcholine.

Embodiment 82 provides the method of any one of Embodiments 78-81,wherein the cationic enzyme substrate is lauroyl choline.

Embodiment 83 provides the method of any one of Embodiments 78-82,wherein the enzyme is acetylcholinesterase.

Embodiment 84 provides a method for detecting a chemical agent, themethod comprising (i) exposing a sensor composition comprising a complexcomprising a p-phenylene ethynylene compound and an enzyme substrate toa sample; (ii) introducing an enzyme to the sensor composition of step(i); and (iii) analyzing the change in fluorescence of the p-phenyleneethynylene compound between the exposing step (i) and the introducing anenzyme step (ii);

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein t is about 1 to about 3; and

the enzyme substrate is lauroyl choline; and

a change in fluorescence between the exposing step (i) and theintroducing an enzyme step (ii) indicates the presence of a chemicalagent that does interact with the enzyme.

Embodiment 85 provides a sensor for detecting the presence of a chemicalagent, the sensor comprising a sensor composition comprising a complexcomprising a p-phenylene ethynylene compound and an enzyme substrate.

Embodiment 86 provides the sensor of Embodiment 85, wherein thep-phenylene ethynylene compound is a cationic p-phenylene ethynylenecompound.

Embodiment 87 provides the sensor of Embodiment 86, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻,            phenolate, (C₁-C₁₀)alkyl-(1,4-substituted            1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and            3-methylimidazolium, and —N⁺(R^(A))₃ wherein at each            occurrence R^(A) is independently (C₁-C₅)alkyl;

j is about 0 to about 4:

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and

m is about 1 to about 10.

Embodiment 88 provides the sensor of any one of Embodiments 86-87,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 89 provides the sensor of any one of Embodiments 86-88,wherein the cationic p-phenylene ethynylene compound has the structure:

wherein s is about 1 to about 3.

Embodiment 90 provides the sensor of any one of Embodiments 86-89,wherein the enzyme substrate is an anionic enzyme substrate.

Embodiment 91 provides the sensor of Embodiment 90, wherein the anionicenzyme substrate is 1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).

Embodiment 92 provides the sensor of any one of Embodiments 85-91,wherein the p-phenylene ethynylene compound is an anionic p-phenyleneethynylene compound.

Embodiment 93 provides the sensor of Embodiment 92, wherein the anionicp-phenylene ethynylene compound has the structure:

wherein

R¹ has the structure:

-   -   wherein        -   at each occurrence Z is independently chosen from —CH₂—,            —O—, —S—, and —NH—;        -   at each occurrence L² is independently chosen from            (C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6,            7, 8, 9, or 10 groups independently chosen from —O—, —S—,            and —NH—;        -   at each occurrence R² is independently chosen from —H,            (C₁-C₅)alkyl, —SO₃ ⁻, —CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and            phenolate;

j is about 0 to about 4:

at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl,

m is about 1 to about 10.

Embodiment 94 provides the sensor of any one of Embodiments 92-93,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein

at each occurrence R³ is independently chosen from —SO₃ ⁻, —CO₂ ⁻,—H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate;

n is about 2 to about 4;

at each occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and

q is about 1 to about 5.

Embodiment 95 provides the sensor of any one of Embodiments 92-94,wherein the anionic p-phenylene ethynylene compound has the structure:

wherein t is about 1 to about 3.

Embodiment 96 provides the sensor of any one of Embodiments 92-95,wherein the enzyme substrate is a cationic enzyme substrate.

Embodiment 97 provides the sensor of any one of Embodiments 92-96,wherein the cationic enzyme substrate is a substituted or unsubstituted(C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.

Embodiment 98 provides the sensor of any one of Embodiments 92-97,wherein the cationic enzyme substrate is a substituted or unsubstituted(C₁-C₂₅)alkyl-C(O)O—(C₁-C₄)alkyl-N⁺(CH₃)₃.

Embodiment 99 provides the sensor of any one of Embodiments 92-98,wherein the cationic enzyme substrate is chosen from lauroyl choline andacetylcholine.

Embodiment 100 provides the sensor of any one of Embodiments 92-99,wherein the cationic enzyme substrate is lauroyl choline.

Embodiment 101 provides a sensor for detecting the presence of achemical agent, the sensor comprising a sensor composition comprising acomplex comprising a p-phenylene ethynylene compound and an enzymesubstrate:

wherein

the p-phenylene ethynylene compound has the structure:

-   -   wherein t is about 1 to about 3; and    -   enzyme substrate is lauroyl choline.

Embodiment 102 provides the method or sensor of any one or anycombination of Embodiments 1-101 optionally configured such that allelements or options recited are available to use or select from.

1-101. (canceled)
 102. A method of inducing germination of microbialspores comprising contacting the microbial spores with a p-phenyleneethynylene compound.
 103. The method of claim 102, wherein thep-phenylene ethynylene compound comprises a repeating unit having thestructure:

wherein R¹ has the structure:

wherein at each occurrence Z is independently chosen from —CH₂—, —O—,—S—, and —NH—; at each occurrence L² is independently chosen from(C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 groups independently chosen from —O—, —S—, and —NH—; at eachoccurrence R² is independently chosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻,—CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, phenolate,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, and—N⁺(R^(A))₃ wherein at each occurrence R^(A) is independently(C₁-C₅)alkyl; at each occurrence L¹ is independently chosen from a bondand

 and j is about 0 to about
 4. 104. The method of claim 102, wherein thep-phenylene ethynylene compound comprises a repeating unit having thestructure:

wherein at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

 and n is about 2 to about
 4. 105. A sensor, the sensor comprising ap-phenylene ethynylene compound and an enzyme substrate.
 106. A methodfor detecting an enzyme, the method comprising (i) introducing an enzymeto the sensor of claim 105; and (ii) analyzing the change influorescence of the p-phenylene ethynylene compound followingintroduction of the enzyme.
 107. The method of claim 106, wherein thep-phenylene ethynylene compound is a cationic p-phenylene ethynylenecompound, wherein the cationic p-phenylene ethynylene compound has thestructure:

wherein R¹ has the structure:

wherein at each occurrence Z is independently chosen from —CH₂—, —O—,—S—, and —NH—; at each occurrence L² is independently chosen from(C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 groups independently chosen from —O—, —S—, and —NH—; at eachoccurrence R² is independently chosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻,—CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, phenolate,(C₁-C₁₀)alkyl-(1,4-substituted1,4-diazabicyclo[2.2.2]octane-1,4-diium)-, and 3-methylimidazolium, and—N⁺(R^(A))₃ wherein at each occurrence R^(A) is independently(C₁-C₅)alkyl; j is about 0 to about 4; at each occurrence L¹ isindependently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl; and m is about 1 to about
 10. 108. Themethod of claim 106, wherein the p-phenylene ethynylene compound is acationic p-phenylene ethynylene compound, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein at each occurrence R³ is independently chosen from —N⁺(CH₃)₃,

n is about 2 to about 4; at each occurrence A is independently chosenfrom —H and —C(O)O—(C₁-C₅)alkyl; and q is about 1 to about
 5. 109. Themethod of claim 106, wherein the p-phenylene ethynylene compound is acationic p-phenylene ethynylene compound, wherein the cationicp-phenylene ethynylene compound has the structure:

wherein t is about 1 to about
 3. 110. The method of claim 106, whereinthe p-phenylene ethynylene compound is an anionic p-phenylene ethynylenecompound, wherein the anionic p-phenylene ethynylene compound has thestructure:

wherein R¹ has the structure:

wherein at each occurrence Z is independently chosen from —CH₂—, —O—,—S—, and —NH—; at each occurrence L² is independently chosen from(C₁-C₅₀)hydrocarbylene interrupted by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 groups independently chosen from —O—, —S—, and —NH—; at eachoccurrence R² is independently chosen from —H, (C₁-C₅)alkyl, —SO₃ ⁻,—CO₂ ⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate; j is about 0 to about4: at each occurrence L¹ is independently chosen from a bond and

at each occurrence A is independently chosen from —H, substituted orunsubstituted —C(O)O—(C₁-C₁₀)hydrocarbyl, and—C(O)NH—(C₁-C₁₀)hydrocarbyl, m is about 1 to about
 10. 111. The methodof claim 106, wherein the p-phenylene ethynylene compound is an anionicp-phenylene ethynylene compound, wherein the anionic p-phenyleneethynylene compound has the structure:

wherein at each occurrence R³ is independently chosen from —SO₃ ⁻, —CO₂⁻, —H₂PO₄ ⁻, HPO₄ ²⁻, PO₄ ³⁻, and phenolate; n is about 2 to about 4; ateach occurrence A is independently chosen from —H and—C(O)O—(C₁-C₅)alkyl; and q is about 1 to about
 5. 112. The method ofclaim 106, wherein the p-phenylene ethynylene compound is an anionicp-phenylene ethynylene compound, wherein the anionic p-phenyleneethynylene compound has the structure:

wherein t is about 1 to about
 3. 113. The method of claim 106, whereinthe enzyme substrate is a cationic enzyme substrate chosen from lauroylcholine and acetylcholine.
 114. The method of claim 106, wherein theenzyme is chosen from phospholipase A1, phospholipase A2, phospholipaseC, and acetyl cholinesterase.
 115. A method for protein analysis, themethod comprising (i) introducing a p-phenylene ethynylene compound to abiological sample comprising at least one protein and (ii) analyzing thefluorescence of the p-phenylene ethynylene compound in the presence ofthe biological sample comprising the at least one protein.
 116. Themethod of claim 115, wherein analyzing the fluorescence of thep-phenylene ethynylene compound in the presence of the biological samplecomprising the at least one protein comprises analyzing the spectralchanges between the fluorescence of the p-phenylene ethynylene compoundof step (i) and the fluorescence of the p-phenylene ethynylene compoundin the presence of the biological sample comprising the at least oneprotein of step (ii).
 117. The method of claim 115, wherein the proteinis at least one of an amyloid beta protein, Aβ-40, Aβ-42, tau, andα-synuclein, islet amyloid precursor protein, Huntingtin, prion,lysozyme, TDP-43 (transactive response DNA-binding protein 43), FUS(fused in sarcoma) and insulin.
 118. A sensor for detecting the presenceof a chemical agent, the sensor comprising a sensor compositioncomprising a complex comprising a p-phenylene ethynylene compound and anenzyme substrate.
 119. A method for detecting a chemical agent, themethod comprising (i) exposing the sensor of claim 118 to a sample; (ii)introducing an enzyme to the sensor composition of step (i); and (iii)analyzing the change in fluorescence of the p-phenylene ethynylenecompound between the exposing step (i) and the introducing an enzymestep (ii).
 120. The method of claim 119, wherein the enzyme substrate isan anionic enzyme substrate that is1,2-dilauroyl-sn-glycero-phospho-(1′-rac-glycerol).
 121. The method ofclaim 119, wherein the p-phenylene ethynylene compound is an anionicp-phenylene ethynylene compound, wherein the enzyme substrate is acationic enzyme substrate, wherein the cationic enzyme substrate is asubstituted or unsubstituted(C₁-C₂₅)hydrocarbyl-C(O)O—(C₁-C₁₀)alkyl-N⁺((C₁-C₅)alkyl)₃.